Antibody-based therapy of transthyretin (ttr) amyloidosis and human-derived antibodies therefor

ABSTRACT

Provided are novel human-derived antibodies specific for transthyretin (TTR), preferably capable of binding misfolded, misassembled, and/or aggregated TTR species, as well as methods related thereto. In addition, methods of diagnosing and/or monitoring diseases and treatments thereof which are associated with TTR amyloidosis are provided. Assays and kits related to anti-bodies specific for TTR or TTR deposits and aggregates are also disclosed. The novel anti-TTR antibodies can be used in pharmaceutical and diagnostic compositions for TTR targeted immunotherapy and diagnostics.

FIELD OF THE INVENTION

The present invention generally relates to antibody-based therapy oftransthyretin (TTR) amyloidosis. In particular, the present inventionrelates to novel molecules specifically binding to human transthyretin(TTR) and antigens thereof, particularly human-derived recombinantantibodies as well as fragments, derivatives and variants thereof thatrecognize the misfolded, misassembled or aggregated forms of TTR or afragments thereof, and which are useful in the treatment of diseases andconditions induced by such pathogenic TTR isoforms.

In addition, the present invention relates to pharmaceutical anddiagnostic compositions comprising such binding molecules, antibodiesand mimics thereof valuable both as a diagnostic tool to identifydiseases associated with TTR amyloidosis and also a passive vaccinationstrategy for treating disorders related to diseases associated with TTRamyloidosis such as Familial Amyloid Polyneuropathy (FAP), FamilialAmyloid Cardiomyopathy (FAC), Senile Systemic Amyloidosis (SSA),systemic familial amyloidosis, leptomeningeal/Central Nervous System(CNS) amyloidosis including Alzheimer disease, TTR-related ocularamyloidosis, TTR-related renal amyloidosis, TTR-relatedhyperthyroxinemia, TTR-related ligament amyloidosis including carpaltunnel syndrome, rotator cuff tears and lumbar spinal stenosis, andpreeclampsia.

Furthermore, the present invention relates to a method of diagnosing adisease or condition induced by pathogenic TTR isoforms, such asmisfolded and/or aggregated TTR present in amyloid deposits, whereinlevels of pathological TTR isoforms are assayed in a sample of a bodyfluid from a subject after administration of an anti-TTR antibody,wherein when compared to a control sample taken before administrationthe presence or alteration in the level of the pathogenic TTR isoforms,for example as determined by the presence of an immuno-complex of TTRand the anti-TTR antibody indicate the disease and/or condition.

BACKGROUND OF THE INVENTION

Transthyretin (TTR), previously named prealbumin, is a soluble proteinof 127 amino-acids (NCBI reference sequence: NP_000362.1) which isinvolved in thyroxin and retinol transport in the body. TTR is secretedin the blood by the liver and in the cerebrospinal fluid by the choroidplexus, and is also expressed in specific tissues like the pancreaticalpha cells or retinal epithelium. TTR synthesis starts at embryonicages and continues during the whole life. It is present at highconcentration in the plasma (3.6-7.2 μM) and CSF (0.04-0.4 μM) andtypically forms under physiological conditions a soluble homotetramer of˜55 kDa.

Under specific conditions which have been poorly elucidated and mayinclude acidic pH, oxidative stress and local factors, the TTR proteinadopts an alternative tridimensional conformation and becomes toxic.

The toxicity of misfolded TTR protein has been discovered byinvestigating a rare, autosomal dominant, neurodegenerative disordernamed Familial Amyloid Polyneuropathy (FAP), which affects adult peoplein their midlife (Plante-Bordeneuve et al., Lancet Neurol. 10 (2011),1086-1097). FAP is characterized by progressive sensory, motor andautonomic impairments leading to death a decade after diagnosis. Nervelesions are associated with the deposition of amorphous aggregates andamyloid fibrils made of TTR protein. The Val30Met substitution is themost frequent mutation causing FAP, especially in areas where thedisease is endemic such as northern Portugal, but more than 100different mutations have been already identified in the TTR gene; seeTable IV below. The pathophysiological mechanism at play is identicalfor all the pathogenic mutations, in that the mutations alter thestructural stability of TTR tetramer, promoting TTR misfolding andleading to the formation of toxic TTR species (Saraiva et al., Curr.Med. Chem. 19 (2012), 2304-2311).

TTR toxicity is also observed as a consequence of the Va1122I1emutation, which is found with high frequency (3-5%) in theAfrican-American and West African populations. This mutation isassociated with Familial Amyloid Cardiomyopathy (FAC), a condition wheremassive TTR accumulation in the myocardium leads to cardiac weaknessesand ultimately cardiac failure (Ruberg et al., Circulation. 126 (2012),1286-1300).

Mutations in the TTR protein sequence are not a strict requirement forTTR toxicity, and the wild-type TTR protein is also prone to misfoldingand formation of toxic aggregates. For example, Senile SystemicAmyloidosis (SSA) is characterized by cardiac weakness and theaccumulation of wild-type TTR aggregates in the myocardium (Ikeda,Amyloid. 18 Suppl 1 (2011), 155-156; Dungu et al., Heart. 98 (2012),1546-1554). Wild-type TTR deposits are also observed in multiple casesof ligament and tendon inflammations including carpal tunnel syndrome,rotator cuff tears and lumbar spinal stenosis (Sueyoshi et al., Hum.Pathol. 42 (2011), 1259-1264; Gioeva et al., Amyloid. 20 (2013), 1-6).Furthermore, TTR amyloidosis has been recently reported in the placentaof mothers suffering from preeclampsia (Kalkunte et al., Am. J. Pathol.183 (2013) 1425-1436).

Treatments for diseases with TTR amyloidosis are limited and mainlyinvasive, wherein primarily the treatment is due to the symptoms. In thecase of FAP, treatments rely on analgesics for the management ofneuropathic pain, on liver transplantation to remove the main source formutated TTR protein, and on treatment with Tafamidis. Tafamidis is asmall molecule which binds to TTR tetramer and stabilizes itsconformation. It acts against the dissociation of the TTR tetramer, therate limiting step in the misfolding pathway leading to the formation oftoxic TTR species. Tafamidis has been approved for the treatment of FAPin Europe but has not been approved in the USA, and its therapeuticefficacy is limited, in the best of cases, to slowing down diseaseprogression. There is currently no treatment available targetingmisfolded TTR protein.

In view of the above, novel therapeutic strategies are needed for anefficacious and safe therapy of diseases associated with TTRamyloidosis.

This technical problem is solved by the embodiments characterized in theclaims and described further below and illustrated in the Examples andFigures.

SUMMARY OF THE INVENTION

The present invention provides anti-transthyretin (TTR) antibodies andequivalent TTR-binding molecules for use in the prophylactic ortherapeutic treatment of diseases and conditions associated with TTRamyloidosis. More specifically, therapeutically useful human-derivedrecombinant antibodies as well as fragments and derivatives thereof thatrecognize misfolded, misassembled or aggregated forms of TTR areprovided.

Misfolded TTR aggregates are associated with markers of cellular stress,oxidative stress, inflammatory response and apoptosis many years beforesymptom onset (Macedo et al., Mol. Med. 13 (2007), 584-91). The naturalcapacity of the body to recognize abnormally folded proteins and degradethem is a protective factor, and differences between patients in theircapacity to eliminate toxic TTR proteins certainly contribute todifferences in age of disease onset and speed of disease progression. Insupport of this hypothesis, it has been shown that patients receiving aliver transplantation from a FAP donor quickly develop antibodiesagainst the pathogenic TTR protein (Ando et al., Transplantation. 73(2002), 751-755), and that FAP patients with high antibody titersagainst the mutated TTR protein have a later disease onset than patientswithout such antibodies (Obayashi et al., Clin. Chim. Acta. 419 (2013),127-131). In addition, active immunization against the pathogenic TTRconformation has been shown to almost completely remove TTR depositionsin FAP transgenic mice (Terazaki et al., Lab. Invest. 86 (2006), 23-31).

However, though it might have seem tempting to investigate animmune-based strategy for therapeutic intervention hitherto the use ofanti-TTR antibodies for the treatment of TTR related diseases has notbeen pursued. For example, in international application WO2010/030203 aparticular isolated mouse monoclonal antibody for TTR has been describedand proposed for use in screening for FAP and in research and treatmentof associated diseases. However, since mouse monoclonal antibodiesinduce human anti-mouse antibody (HAMA) response they are not suitablefor therapy in human. Hence, since the international application lapsedand no subsequent development published yet, apparently a therapeuticantibody-based approach has not been followed. Rather, so far foranti-TTR antibodies only their diagnostic utility for patients with TTRamyloidosis has been further investigated; see, e.g., Phay M. et al.,Rejuvenation Res. 2013 Oct. 28. [Epub ahead of print].

In contrast, experiments performed in accordance with the presentinvention were successful in the isolation of human-derived monoclonalTTR-specific antibodies which maturated in the human body and arespecific for misfolded, misassembled, mutated, and/or aggregated TTRspecies and/or fragments thereof. The human subjects and patients,respectively, being the source of the B cells from which thehuman-derived monoclonal anti-TTR antibodies and the cDNA encoding theirvariable domain, respectively, have been isolated, did not show asubstantial amount of misfolded TTR and were symptom-free of conditionsassociated with pathogenic isoforms. However, in another embodiment ofthe present invention, the source of the B cells from which thehuman-derived monoclonal anti-TTR antibodies and the cDNA encoding theirvariable domain, respectively, might be isolated are patients showingsymptoms of a disease and/or disorder associated with TTR amyloidosis.Therefore, it is prudent to expect that the human monoclonal anti-TTRantibodies of the present invention and derivatives thereof besidesbeing non-immunogenic in human exhibit a therapeutically beneficialeffect.

The present invention is thus directed to human-derived recombinantantibodies, antigen-binding fragments and similar antigen-bindingmolecules which are capable of specifically recognizing TTR. If notindicated otherwise, by “specifically recognizing TTR”, “antibodyspecific to/for TTR” and “anti-TTR antibody” antibodies are meant whichspecifically, generally, and collectively binds to the native monomericform of TTR; antibodies binding specifically to either forms of TTR,e.g.

mutated TTR, oligomeric, fibrillar and/or non-fibrillar TTR. Providedherein are human-derived antibodies selective for full-length and/orfragments and/or misfolded, misassembled and/or aggregated forms of TTR.

As mentioned before, preferably the anti-TTR antibody of the presentinvention is a recombinant antibody, wherein at least one, preferablytwo or more preferably all three complementarity determining regions(CDRs) of the variable heavy and/or light chain, and/or substantiallythe entire variable region are encoded by a cDNA derived from an mRNAobtained from a human memory B cell which produced an anti-TTR antibody.In a preferred embodiment, the anti-TTR antibody of the presentinvention displays, in any combination one more of the binding andbiological properties as demonstrated for the subject antibodiesillustrated in the appended Examples and Figures, preferably one more ofthe binding and biological properties as demonstrated for exemplaryantibodies NI-301.59.F1, NI-301.35G11, and NI-301.37F1.

In a particularly preferred embodiment of the present invention, theanti-TTR antibody or TTR-binding fragment thereof demonstrates theimmunological binding characteristics of an antibody characterized bythe variable regions VH and/or VL as set forth in FIG. 1.

The antigen-binding fragment of the antibody can be a single chain Fvfragment, an F(ab′) fragment, an F(ab) fragment, and an F(ab′)2fragment, or any other antigen-binding fragment. In a specificembodiment, infra, the antibody or fragment thereof is a human IgGisotype antibody. Alternatively, the antibody is a chimeric human-rodentor rodentized antibody such as murine or murinized, rat or ratinizedantibody, the rodent versions being particularly useful for diagnosticmethods and studies in animals.

Furthermore, the present invention relates to compositions comprisingthe antibody of the present invention or active fragments thereof and toimmunotherapeutic and immunodiagnostic methods using such compositionsin the prevention, diagnosis or treatment of disorders associated withTTR amyloidosis, wherein an effective amount of the composition isadministered to a patient in need thereof.

The present invention also relates to polynucleotides encoding at leasta variable region of an immunoglobulin chain of the antibody of theinvention. Preferably, said variable region comprises at least onecomplementarity determining region (CDR) of the V_(H) and/or V_(L) ofthe variable region as set forth in FIG. 1. In a preferred embodiment ofthe present invention, the polynucleotide is a cDNA, preferably derivedfrom mRNA obtained from human memory B cells which produce antibodiesreactive with mutant, misfolded, misassembled and/or aggregated TTRspecies.

Accordingly, the present invention also encompasses vectors comprisingsaid polynucleotides and host cells transformed therewith as well astheir use for the production of an antibody and equivalent bindingmolecules which are specific for TTR. In a further embodiment of thepresent invention, the antibodies or binding molecules are capable ofbinding misfolded, misassembled or aggregated TTR species or fragmentsthereof Means and methods for the recombinant production of antibodiesand mimics thereof as well as methods of screening for competing bindingmolecules, which may or may not be antibodies, are known in the art.However, as described herein, in particular with respect to therapeuticapplications in human the antibody of the present invention is a humanantibody in the sense that application of said antibody is substantiallyfree of an immune response directed against such antibody otherwiseobserved for chimeric and even humanized antibodies.

Furthermore, disclosed herein are compositions and methods that can beused to identify TTR, in particular mutated, misfolded, misassembled, oraggregated TTR species or fragments in samples and/or in vivo. Thedisclosed anti-TTR antibodies and binding fragments thereof can be usedto screen human blood, plasma, serum, saliva, peritoneal fluid,cerebrospinal fluid (“CSF”), and urine for the presence of TTR and/ormutated, misfolded, misassembled, or aggregated TTR species or fragmentsthereof in samples, for example, by using ELISA-based or surface adaptedassay. In one embodiment the present invention relates to a method ofdiagnosing or monitoring the progression of a disorder related tomutated, misfolded, misassembled, or aggregated TTR species or fragmentsthereof in a subject, the method comprising determining the presence ofmutated, misfolded, misassembled, or aggregated TTR species or fragmentsin a sample from the subject to be diagnosed with at least one antibodyof the present invention or an TTR binding molecule and/or bindingmolecules for misfolded, misassembled, or aggregated TTR species orfragments having substantially the same binding specificities of any onethereof, wherein the presence of misfolded, misassembled, or aggregatedTTR species or fragments is indicative of the disorder.

Furthermore, in one embodiment of the present invention the anti-TTRantibodies and TTR-binding molecules comprising at least one CDR of anantibody of the present invention are provided for the preparation of acomposition for in vivo detection (also called in vivo imaging) of ortargeting a therapeutic and/or diagnostic agent to TTR, in particularmutated, misfolded, misassembled, or aggregated TTR species or fragmentsin the human or animal body. The methods and compositions disclosedherein can aid in disorders associated with TTR amyloidosis andcharacterized, e.g., by the occurrence of forms of TTR and can be usedto monitor disease progression and therapeutic efficacy of the therapyprovided to the subject, for example in in vivo imaging relateddiagnostic methods. Therefore, in one embodiment the anti-TTR antibodyand/or TTR binding molecule of the present invention is provided,wherein said in vivo detection (imaging) comprises scintigraphy,positron emission tomography (PET), single photon emission tomography(SPECT), near infrared (NIR) optical imaging or magnetic resonanceimaging (MRI).

Hence, it is a particular object of the present invention to providemethods for treating, diagnosing or preventing a disease associated withTTR amyloidosis. The methods comprise administering an effectiveconcentration of a preferably human antibody or antibody derivative tothe subject where the antibody targets TTR or fragments thereof,preferably misfolded, misassembled, or aggregated TTR species orfragments thereof

In a further aspect the present invention provides a peptide having anepitope of TTR, preferably of misfolded, misassembled, or aggregated TTRspecies or fragments thereof specifically recognized by an antibody ofthe present invention. Said peptide comprises or consists of an aminoacid sequence as indicated below in the detailed description and in theexamples or a modified sequence thereof in which one or more amino acidsare substituted, deleted and/or added, with the proviso that the peptideis still recognized by the cognate antibody. As mentioned, such peptidecan be used as an antigen, i.e. being an immunogen and thus useful foreliciting an immune response in a subject and stimulating the productionof an antibody of the present invention in vivo. Accordingly, thepeptide of the present invention is particularly useful as a vaccine.

Additionally, the present invention provides a method for diagnosingdiseases associated with TTR amyloidosis in a subject, comprising a stepof determining the presence of an antibody that binds to said peptide ina biological sample of said subject.

In a further aspect, the present invention relates to a method ofdiagnosing a disease associated with TTR amyloidosis, monitoring thetreatment of the disease with an anti-TTR antibody or determining thediagnostic or therapeutic utility of an anti-TTR antibody comprisingassaying the level of misfolded and/or aggregated TTR in a sample, forexample blood obtained from a subject following administration of ananti-TTR antibody to the subject, wherein the presence or elevated thelevel of misfolded and/or aggregated TTR in the sample of the subjectcompared to the control such as a sample obtained from the subject priorto administration of the anti-TTR antibody indicates a diseaseassociated with TTR amyloidosis.

In one preferred embodiment of the present invention, in particular whenusing non-human animals for testing recombinant human-derived antibodiesas illustrated in Example 13 and other anti-TTR antibodies intended foruse in humans in general the level of misfolded and/or aggregated TTR inthe sample is assayed by determining a complex formed between theanti-TTR antibody and the misfolded and/or aggregated TTR, for exampleby immuno-precipitation with an anti-human IgG or anti-idiotypicantibody.

With respect to the diagnostic aspect in particular for a human subjectand patient, the presence and elevated level of misfolded and/oraggregated TTR and complex thereof with the anti-TTR antibody,respectively, indicates the presence of TTR amyloid deposits in thehuman body, for example in the heart, peripheral nervous system (PNS),eyes, muscles, gastro-intestinal tract, kidneys, vascular system and thecentral nervous system (CNS) of a patient or subject. Thus, the methodof the present invention allows the identification and determination ofa disease associated with TTR amyloidosis in the subject body on the onehand and removal of TTR deposits from patient's body on the other,thereby also indicating the therapeutic progress of a given treatmentand efficacy of a drug for the treatment of TTR amyloidosis such as ananti-TTR antibody.

Hence, as demonstrated in Example 13 the anti-TTR antibody of thepresent invention is capable of binding misfolded and/or aggregated TTRwith sufficient affinity to alter the stability of pathological TTRdeposits such as to capture and remove misfolded and/or aggregated TTRfrom the deposits into a body fluid, in particular blood. The specifiedtime interval following administration, i.e. the time frame after whichthe level of pathological TTR and complex with the anti-TTR antibody,respectively, is measured is determined by a practicing physician.Normally, a time interval less than a week is used. In a preferredembodiment, the level of pathological TTR in a sample from a patient orsubject after administration of an anti-TTR antibody or antigen-bindingfragment thereof to the patient or subject is determined after less thanor equal to 48 hours; see also Example 13.

The present invention also relates to the use of any anti-TTR antibodyand TTR-binding molecule in the method described above. However, due tothe advantageous properties and in particular because beinghuman-derived the use of an anti-TTR antibody of the present disclosedherein is preferred. In a preferred embodiment, the antibody showssubstantially the same binding and biological activities as any antibodyselected from NI-301.59F1, NI-301.35G11, NI-301.37F1, NI-301.2F5,NI-301.28B3, NI-301.119C12, NI-301.5D8, NI-301.9D5, NI-301.104F5,NI-301.21F10, NI-301.9G12, NI-301.12D3, NI-301.37F1-PIMC, NI-301.44E4,NI-301.18C4, NI-301.11A10, NI-301.3C9, NI-301.14D8, NI-301.9X4, andNI-301.14C3. The anti-TTR antibody can also be altered to facilitate thehandling of the method of diagnosing including the labeling of theantibody as described in detail below.

Further embodiments of the present invention will be apparent from thedescription and Examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Amino acid sequences of the variable regions of human antibodiesNI-301.59F1, NI-301.35G11, NI-301.37F1, NI-301.2F5, NI-301.28B3,NI-301.119C12, NI-301.5D8, NI-301.9D5, NI-301.104F5, NI-301.21F10,NI-301.9G12, NI-301.12D3, NI-301.37F1-PIMC, NI-301.44E4, NI-301.18C4,NI-301.11A10, NI-301.3C9, NI-301.14D8, NI-301.9X4, and NI-301.14C3.

-   -   Framework (FR) and complementarity determining regions (CDRs)        are indicated with the CDRs being underlined. The Kabat        numbering scheme was used (cf. http://www.bioinforg.uk/abs/).

FIG. 2: Binding to aggregated, wild-type and mutant TTR by direct ELISA.

-   -   A, B, C: ELISA plates were coated with aggregated human        wild-type TTR (), aggregated recombinant V30M-TTR (▾) and        bovine serum albumin (BSA) (▪) at 10 μg/ml, and incubated with        the following human monoclonal antibodies at a concentration        range from 4 pM to 400 nM: A) NI-301.59F1, B) NI-301.35G11        and C) NI-301.37F1.    -   EC50 values were estimated by fitting data points with the least        square method.    -   NI-301.59F1: aggregated wt-TTR EC₅₀=3.0 nM, aggregated V30M-TTR        EC₅₀=15.5 nM NI-301.35G11: aggregated wt-TTR EC₅₀=3.9 nM,        aggregated V30M-TTR EC₅₀=5.0 nM NI-301.37F1: aggregated wt-TTR        EC₅₀=0.35 nM, aggregated V30M-TTR EC₅₀=0.15 nM

FIG. 3: Specificity for aggregated TTR on dot blot.

-   -   Human wild-type TTR protein in native (1) or aggregated (2)        conformations, and aggregated recombinant V30M-TTR protein (3)        were deposited on a nitrocellulose membrane and incubated with        the following antibodies: A) commercial rabbit polyclonal        antibody against TTR (Dako-A0002; 150 ng/ml), B) NI-301.59F1 C)        NI-301.35G11 and D) NI-301.37F1 (B, C and D: human monoclonal        antibodies at 50 nM).

FIG. 4: Specificity for aggregated TTR on western blot.

-   -   Human wild-type TTR protein (300 ng) in native (1) or        aggregated (2) conformations, and wild-type mouse liver extract        (10 μg total protein) (3) were loaded on a SDS-PAGE gel and        processed for western-blot with the following antibodies: A)        commercial rabbit polyclonal antibody against TTR (Dako-A0002;        150 ng/ml), B) NI-301.59F1 C) NI-301.35G11 and D) NI-301.37F1        (B, C and D: human monoclonal antibodies at 50 nM). To prevent        dissociation of the high molecular weight aggregates, the        aggregated TTR sample was crosslinked with glutaraldehyde (1%, 5        min) prior to loading on the gel.

FIG. 5: Absence of binding to human plasma TTR on western blot.

-   -   Plasma samples (0.5 μl) from controls (n=5), asymptomatic        mutation carriers (n=5) and FAP patients (n=4) were loaded on a        SDS-PAGE gel and processed for western blot with the following        antibodies: A) commercial rabbit polyclonal antibody against TTR        (Dako-A0002; 150 ng/ml), B) secondary antibody only (anti-human        IgG-HRP, 1/10 000 dilution), C) NI-301.35G11 and D) NI-301.37F1        (C and D: human monoclonal antibodies at 50 nM).

FIG. 6: Absence of binding to human plasma TTR on dot blot.

-   -   Pure wild-type and mutant TTR protein in native and aggregated        conformations, and plasma samples from controls, asymptomatic        mutation carriers and FAP patients were deposited on a        nitrocellulose membrane and incubated with the following        antibodies: A) commercial rabbit polyclonal antibody against TTR        (Dako-A0002; 150 ng/ml), B) secondary antibody only (anti-mouse        IgG2a-HRP, 1/10 000 dilution), and C) mouse chimeric antibody        NI-301.mur35G11 (10 nM).    -   Samples 1-6: 150 ng of 1) aggregated wt-TTR, 2) native        wt-TTR, 3) BSA, 4) native V30M-TTR, 5) native L55P-TTR and 6)        native Y78F-TTR.    -   Samples 7-18: 2 μl of plasma collected from 7-10) controls        (n=4), 11-14) asymptomatic mutation carriers (n=4) and 15-18)        FAP patients (n=4).

FIG. 7: Specific binding to aggregated TTR in solution.

-   -   Human wild-type and recombinant TTR protein in native and        aggregated conformations, and a human plasma sample at 3        different dilutions were used for TTR immunoprecipitation (IP)        using the following antibodies: A) commercial rabbit polyclonal        antibody against TTR (Dako-A0002), B) NI-301.35G11 and C)        NI-301.37F1. The immunoprecipitated proteins were submitted to        SDS-PAGE and detected by western blot (WB) with the Dako-A0002        antibody (150 ng/ml).    -   Lanes 1-2: WB loading controls: 300 ng of 1) human wt-TTR, 2)        recombinant wt-TTR Lanes 3-6: IP on pure TTR protein: 3) human        native wt-TTR, 4) human aggregated wt-TTR, 5) recombinant native        wt-TTR and 6) recombinant aggregated wt-TTR    -   Lanes 7-10: IP on human plasma diluted 7) 10 times, 8) 100        times, 9) 1000 times with PBS, and 10) PBS only

FIG. 8: Specific binding to TTR on FAP mouse tissue.

-   -   Transgenic mice expressing the human V30M-TTR allele on a TTR        knock-out (KO) background reproduce the histopathological        hallmarks of FAP, including amorphous and amyloid TTR deposits        in various tissues. Liver and intestine tissue sections        collected from A) FAP mice and B) TTR-KO mice were processed for        immunohistochemistry using the following antibodies: 1)        commercial rabbit polyclonal antibody against TTR (Dako-A0002;        1/1000 dilution), 2) NI-301.35G11 and 3) NI-301.37F1 (2 and 3:        human monoclonal antibodies at 50 nM).

FIG. 9: Specific binding to misfolded TTR deposits but not native TTR inhuman tissue.

-   -   Antibodies were characterized for their capacity to bind TTR on        sections from FAP patient skin biopsy and healthy control        pancreas: the misfolded TTR accumulations that are        characteristic for FAP are present in the patient skin biopsy,        whereas pancreatic alpha cells show endogenous expression of        TTR. Sections were processed for immunohistochemistry using the        following antibodies: 1A) commercial rabbit polyclonal antibody        against TTR (Dako-A0002; 1/1000 dilution), 1B) HRP-coupled        anti-rabbit IgG antibody ( 1/125 dilution), 2A) mouse chimeric        antibody NI-301.mur35G11 (50 nM), 2B) HRP-coupled anti-mouse        IgG2a antibody ( 1/125 dilution), 3A) NI-301.37F1 (50 nM), and        3B) HRP-coupled anti-human IgG ( 1/125 dilution).

FIG. 10: TTR binding epitopes assessed by pepscan analysis.

-   -   The antibody binding epitopes on TTR were determined using the        peptide scan method. In addition to the peptides covering the        full human wild-type TTR sequence (spots 1 to 29), selected TTR        mutations were also represented on the membrane (spots 30 to        44). The peptide scan membrane was incubated with the following        antibodies at 50 nM: A) NI-301.59F1, B) NI-301.35G11 and C)        NI-301 .37F1. As summarized in Table D): NI-301.59F1 binds        EEEFVEGIY (TTR 61-69); NI-301.35G11 binds GELHGLTTEEE (TTR        53-63); the L55P mutation prevents antibody binding; and    -   NI-301.37F1 binds WEPFA (TTR 41-45); the E42G mutation prevents        antibody binding. In order to determine the sequence        requirements of the mentioned epitopes, the antibody binding        epitopes on TTR were further identified using the alanine scan        method. The whole sequence of human wild-type TTR protein was        represented on the membrane as a set of 151 successive peptides        of 15 amino-acids in length, starting at every amino-acid of the        TTR protein. For each peptide, the amino-acid in position 10 was        replaced by an alanine, or by glycine or proline when the        initial amino-acid was an alanine. The peptide scan membrane was        incubated with the following antibodies at 20 nM: E)        NI-301.59F1, F) NI-301.35G11 and G) NI-301.37F1. As summarized        in Table H):    -   NI-301.59F1 binds EEFXEGIY (TTR 61-68).    -   NI-301.35G11 binds ELXGLTXE (TTR 54-61).    -   NI-301.37F1 binds WEPFA (TTR 41-45), wherein X denotes amino        acid; Replacement of E42 by alanine did not disrupt binding but        replacement by guanine prevented antibody binding as reported in        C.

FIG. 11: Antibody binding kinetics to TTR protein in solution assessedby surface plasmon resonance.

-   -   The binding kinetics of antibody NI-301.37F1 to TTR protein was        measured by surface plasmon resonance (SPR). Antibody        NI-301.37F1 was captured on the sensor by means of an anti-human        IgG antibody, and TTR protein solution was flown over the sensor        surface, at concentrations ranging from 3.2 to 316 nM. A simple        1:1 binding model was used to fit the data and derive the        respective association (ka) and dissociation (kd) constants and        the affinity (KD). Binding properties were determined for A)        human wild-type TTR protein in native conformation, B)        denaturated, human wild-type TTR protein (misfolded        conformation), and C) recombinant mutant TTR-L55P protein.    -   Native wild-type TTR: ka=not determined, kd=not determined,        KD>316 nM    -   Denaturated wild-type TTR: ka=2.1 104 M-1s-1, kd=2.6 10-5 s-1,        KD=1.2 nM    -   Recombinant TTR-L55P: ka=3.3 104 M-1s-1, kd=4.6 10-5 s-1, KD=1.4        nM

FIG. 12: Chronic treatment with anti-TTR antibody reduces pathologicalTTR deposition in FAP mouse model.

-   -   FAP mice (Tg(6.0hMet30)×muTTR-KO) received weekly administration        of mouse chimeric NI-301.37F1 or isotype control antibody at 3        mg/kg i.p. for 12 weeks. At the end of the treatment period,        tissues were collected and the extent of TTR deposition was        quantified by immunofluorescence. A) Effect of treatment in        7-month old mice (n=14-15 mice per group); B) Effect of        treatment in 17-month old mice (n=10 mice per group). Group        comparisons with two tailed, unpaired t-test.

FIG. 13: Antibody binding to pathological TTR deposits in vivo.

-   -   Target engagement was characterized in adult FAP mice (7 months)        48 hours after administration of a single dose of antibody        NI-301.37F1 at 30 mg/kg i.p, or PBS. Pathological TTR deposits        and localization of the injected antibody were detected        simultaneously by immunofluorescence.    -   A, D) Pathological TTR deposits in the kidneys of (A)        NI-301.37F1- or (D) PBS-injected mice. B, E) Detection of human        antibody in (B) NI-301.37F1- or (E) PBS-injected mice. C, F)        Overlayed images showing TTR and NI-301.37F1 (C) colocalization        and (F) absence of unspecific staining.

FIG. 14: Tissue-free detection of misfolded TTR in vivo.

-   -   Adult FAP mice received a single administration of NI-301.37F1        or isotype control antibody at 3 mg/kg i.p. Blood samples were        collected prior antibody injection (t=0) and 48 hours after        antibody injection (t=48 h). Plasma samples were processed by        immunoprecipitation with an anti-human IgG antibody, and        analyzed by western blot using for detection: A) a        conformation-independent, anti-TTR polyclonal antibody (Dako        A0002, 150 ng/ml), and B) NI-301.37F1 (20 nM). In parallel, a        plasma sample obtained from an uninjected FAP mouse was        incubated with antibody NI-301.37F1 in vitro, before processing.

FIG. 15: Antibody specificity evaluated against aggregating proteins byELISA

-   -   Antibody specificity for TTR protein was evaluated by measuring        binding to selected aggregating proteins by direct ELISA.        Antibody binding was evaluated at 4 and 20 nM and signal        intensity was expressed in fold change relative to background        levels, measured for each assay in absence of anti-TTR antibody.    -   A-B) NI-301.37F1 binding assayed at 20 (A) and 4 nM (B)    -   C-D) NI-301.44E4 binding assayed at 20 (C) and 4 nM (D).

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to immunotherapy andnon-invasive methods for the detection of diseases and conditionsassociated with the presence of pathologic, often mutant and/ormisfolded isoforms of transthyretin (TTR). More specifically, thepresent invention relates to recombinant human-derived monoclonalantibodies and antigen binding fragments thereof, which have beengenerated based on sequence information obtained from selected humandonor populations and are capable of binding to such TTR isoforms andantigens thereof The recombinant human-derived monoclonal antibody ofthe present invention is advantageously characterized by specificallybinding to misfolded, misassembled, mutated, and/or aggregated TTRspecies and/or fragments thereof allowing a targeting for treatmentand/or diagnosis ofpathological altered TTR species. Due to their humanderivation, the resulting recombinant antibodies of the presentinvention can be reasonably expected to be efficacious and safe astherapeutic agent, and highly specific as a diagnostic reagent for thedetection of pathological TTR without giving false positives.

In addition, the antibody of the present invention as well as thederivatives thereof can be used for combination therapy of patientsafter organ transplantations who nevertheless bear the risk ofdeveloping a TTR amyloidosis due to e.g. their deposition, e.g.inheritable mutations in the TTR or a defect in the production of TTR inthe liver. Thus, as a particular advantageous embodiment, the presentinvention relates to the human monoclonal antibody and any derivativesthereof described herein for use in the treatment of patients eitheralone or in the treatment of patients receiving e.g. immunosuppressivedrugs after organ transplantation or other agents utilized for symptomsassociated with TTR amyloidosis, wherein the antibody of the presentinvention and any of its derivatives is designed to be administeredconcomitantly with the immunosuppressive drug and/or the agentsuppressing further side effects or sequentially before or afteradministration of the same. In this context, the anti-TTR antibody andTTR-binding fragment ofthe present invention are preferablysubstantially non-immunogenic in human. In one embodiment of the presentinvention, pharmaceutical compositions are provided comprising both ahuman monoclonal antibody of the present invention or any derivativesthereof and one or more immunosuppressive drugs and/or utilized forsymptoms associated with TTR amyloidosis.

I. Definitions

Unless otherwise stated, a term as used herein is given the definitionas provided in the Oxford Dictionary of Biochemistry and MolecularBiology, Oxford University Press, 1997, revised 2000 and reprinted 2003,ISBN 0 19 850673 2.

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “an antibody,” is understood to representone or more antibodies. As such, the terms “a” (or “an”), “one or more,”and “at least one” can be used interchangeably herein.

If not specifically indicated otherwise, the term “TTR”, is usedinterchangeably to specifically refer to the different forms oftransthyretin (TTR). The term “TTR” is also used to generally identifyother conformers of TTR, for example, oligomers and/or misfolded,misassembled and/or aggregated forms of TTR. The term “TTR” is also usedto refer collectively to all types and forms of TTR, such as mutatedTTR. Added letters in front of the terms TTR are used to indicate theorganism the particular ortholog is originating from, e.g. hTTR forhuman TTR or mTTR for murine origin. In addition, unless indicatedotherwise the numbering system for TTR amino acid sequence used hereinrefers to the mature TTR protein, i.e. the TTR protein as secreted bythe cells after cleavage of the signal peptide. This numbering is theone used to define TTR mutations found in patients, such as TTR-V30M orTTR-L55P, but differs from the one used for transthyrethin precursorprotein sequence (NCBI reference sequence: NP_000362.1). In thiscontext, the position and substituted amino acid in a mutant TTR may beindicated in different but equivalent ways; see, e.g., “TTR-V30M” and“V30M-TTR”.

The anti-TTR antibodies disclosed herein specifically bind TTR andepitopes thereof and to various conformations of TTR and epitopesthereof. For example, disclosed herein are antibodies that specificallybind pathologically altered TTR species or fragments thereof, such asoligomers/fibrils and/or mutated, misfolded, misassembled and/oraggregated forms of TTR or fragments thereof The term (pathologically)mutated, misfolded, misassembled aggregated/aggregates of TTR is usedinterchangeable to specifically refer to the aforementioned forms. Theterm (pathological) “aggregated forms” or “aggregates” as used hereindescribes the products of an accumulation or cluster formation due toTTR erroneous/pathological interaction with one another. Theseaggregates, accumulations or cluster forms may be, substantially consistor consist of both TTR and/or TTR fragments and of non-fibrillaroligomers and/or fibrillar oligomers and fibrils thereof As used herein,reference to an antibody that “specifically binds”, “selectively binds”,or “preferentially binds” TTR refers to an antibody that does not bindother unrelated proteins. In one example, a TTR antibody disclosedherein can bind TTR or an epitope thereof and show no binding aboveabout 2 times background for other proteins. In a preferred embodiment,the antibody of the present invention does not substantially recognizeunrelated amyloid-forming proteins selected from the group consisting ofalpha-synuclein (a-syn), Tau, transactive response DNA binding protein43 (TDP-43), serum amyloid A (SAA), huntingtin protein (HTT); see, e.g.FIG. 15. An antibody that “specifically binds” or “selectively binds” aTTR conformer refers to an antibody that does not bind all conformationsof TTR, i.e., does not bind at least one other TTR conformer.

For example, disclosed herein are antibodies that can preferentiallybind to misfolded, misassembled and/or aggregated forms of TTR both invitro and in tissues obtained from patients with diseases associatedwith TTR amyloidosis or with a risk to develop diseases associated withTTR amyloidosis. Since the sequences of the TTR antibodies of thepresent invention have been obtained from human subjects, the TTRantibodies of the present invention may also be called “humanauto-antibodies” or “human-derived antibodies” in order to emphasizethat those antibodies were indeed expressed initially by the subjectsand are not synthetic constructs generated, for example, by means ofhuman immunoglobulin expressing phage libraries, which hithertorepresented one common method for trying to provide human-likeantibodies.

The term “peptide” is understood to include the terms “polypeptide” and“protein” (which, at times, may be used interchangeably herein) withinits meaning. Similarly, fragments of proteins and polypeptides are alsocontemplated and may be referred to herein as “peptides”. Nevertheless,the term “peptide” preferably denotes an amino acid polymer including atleast 5 contiguous amino acids, preferably at least 10 contiguous aminoacids, more preferably at least 15 contiguous amino acids, still morepreferably at least 20 contiguous amino acids, and particularlypreferred at least 25 contiguous amino acids. In addition, the peptidein accordance with present invention typically has no more than 100contiguous amino acids, preferably less than 80 contiguous amino acids,more preferably less than 50 contiguous amino acids and still morepreferred no more than 15 contiguous amino acids of the TTR polypeptide.

Polypeptides:

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, “peptides,” “dipeptides,”“tripeptides, “oligopeptides,” “protein,” “amino acid chain,” or anyother term used to refer to a chain or chains of two or more aminoacids, are included within the definition of “polypeptide,” and the term“polypeptide” may be used instead of, or interchangeably with any ofthese terms.

The term “polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation andderivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide may be derived from a natural biological source or producedby recombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It may be generated in any manner,including by chemical synthesis.

A polypeptide of the invention may be of a size of about 3 or more, 5 ormore, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 ormore, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides may have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides which do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, and arereferred to as unfolded. As used herein, the term glycoprotein refers toa protein coupled to at least one carbohydrate moiety that is attachedto the protein via an oxygen-containing or a nitrogen-containing sidechain of an amino acid residue, e.g., a serine residue or an asparagineresidue.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated for purposed of the invention, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

“Recombinant peptides, polypeptides or proteins” refer to peptides,polypeptides or proteins produced by recombinant DNA techniques, i.e.produced from cells, microbial or mammalian, transformed by an exogenousrecombinant DNA expression construct encoding the fusion proteinincluding the desired peptide. Proteins or peptides expressed in mostbacterial cultures will typically be free of glycan. Proteins orpolypeptides expressed in yeast may have a glycosylation patterndifferent from that expressed in mammalian cells.

Included as polypeptides of the present invention are fragments,derivatives, analogs or variants of the foregoing polypeptides and anycombinations thereof as well. The terms “fragment,” “variant,”“derivative”, and “analog” include peptides and polypeptides having anamino acid sequence sufficiently similar to the amino acid sequence ofthe natural peptide. The term “sufficiently similar” means a first aminoacid sequence that contains a sufficient or minimum number of identicalor equivalent amino acid residues relative to a second amino acidsequence such that the first and second amino acid sequences have acommon structural domain and/or common functional activity. For example,amino acid sequences that comprise a common structural domain that is atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99%, or at least about 100%, identical are definedherein as sufficiently similar. Preferably, variants will besufficiently similar to the amino acid sequence of the preferredpeptides of the present invention, in particular to TTR, variants,derivatives or analogs of either of them. Such variants generally retainthe functional activity of the peptides of the present invention.Variants include peptides that differ in amino acid sequence from thenative and wt peptide, respectively, by way of one or more amino aciddeletion(s), addition(s), and/or substitution(s). These may be naturallyoccurring variants as well as artificially designed ones.

Furthermore, the terms “fragment,” “variant,” “derivative”, and “analog”when referring to antibodies or antibody polypeptides of the presentinvention include any polypeptides which retain at least some of theantigen-binding properties of the corresponding native binding molecule,antibody, or polypeptide. Fragments of polypeptides of the presentinvention include proteolytic fragments, as well as deletion fragments,in addition to specific antibody fragments discussed elsewhere herein.Variants of antibodies and antibody polypeptides of the presentinvention include fragments as described above, and also polypeptideswith altered amino acid sequences due to amino acid substitutions,deletions, or insertions. Variants may occur naturally or benon-naturally occurring. Non-naturally occurring variants may beproduced using art-known mutagenesis techniques. Variant polypeptidesmay comprise conservative or non-conservative amino acid substitutions,deletions or additions. Derivatives of TTR specific binding molecules,e.g., antibodies and antibody polypeptides of the present invention, arepolypeptides which have been altered so as to exhibit additionalfeatures not found on the native polypeptide. Examples include fusionproteins. Variant polypeptides may also be referred to herein as“polypeptide analogs”. As used herein a “derivative” of a bindingmolecule or fragment thereof, an antibody, or an antibody polypeptiderefers to a subject polypeptide having one or more residues chemicallyderivatized by reaction of a functional side group. Also included as“derivatives” are those peptides which contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acids. Forexample, 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine.

Determination of Similarity and/or Identity of Molecules: “Similarity”between two peptides is determined by comparing the amino acid sequenceof one peptide to the sequence of a second peptide. An amino acid of onepeptide is similar to the corresponding amino acid of a second peptideif it is identical or a conservative amino acid substitution.Conservative substitutions include those described in Dayhoff, M. O.,ed., The Atlas of Protein Sequence and Structure 5, National BiomedicalResearch Foundation, Washington, D.C. (1978), and in Argos, EMBO J. 8(1989), 779-785. For example, amino acids belonging to one of thefollowing groups represent conservative changes or substitutions: -Ala,Pro, Gly, Gln, Asn, Ser, Thr; -Cys, Ser, Tyr, Thr; -Val, Ile, Leu, Met,Ala, Phe; -Lys, Arg, His; -Phe, Tyr, Trp, His; and -Asp, Glu.

“Similarity” between two polynucleotides is determined by comparing thenucleic acid sequence of one polynucleotide to the sequence of apolynucleotide. A nucleic acid of one polynucleotide is similar to thecorresponding nucleic acid of a second polynucleotide if it is identicalor, if the nucleic acid is part of a coding sequence, the respectivetriplet comprising the nucleic acid encodes for the same amino acid orfor a conservative amino acid substitution.

The determination of percent identity or similarity between twosequences is preferably accomplished using the mathematical algorithm ofKarlin and Altschul (1993) Proc. Natl. Acad. Sci USA 90: 5873-5877. Suchan algorithm is incorporated into the BLASTn and BLASTp programs ofAltschul et al. (1990) J. Mol. Biol. 215: 403-410 available at NCBI(http://www.ncbi.nlm.nih.gov/blast/Blast.cge).

The determination of percent identity or similarity is performed withthe standard parameters of the BLASTn programs for BLAST polynucleotidesearches and BLASTp programs for BLAST protein search, as recommended onthe NCBI webpage and in the “BLAST Program Selection Guide” in respectof sequences of a specific length and composition.

BLAST polynucleotide searches are performed with the BLASTn program. Forthe general parameters, the “Max Target Sequences” box may be set to100, the “Short queries” box may be ticked, the “Expect threshold” boxmay be set to 1000 and the “Word Size” box may be set to 7 asrecommended for short sequences (less than 20 bases) on the NCBIwebpage. For longer sequences the “Expect threshold” box may be set to10 and the “Word Size” box may be set to 11. For the scoring parametersthe “Match/mismatch Scores” may be set to 1,−2 and the “Gap Costs” boxmay be set to linear. For the Filters and Masking parameters, the “Lowcomplexity regions” box may not be ticked, the “Species-specificrepeats” box may not be ticked, the “Mask for lookup table only” box maybe ticked, the “DUST Filter Settings” may be ticked and the “Mask lowercase letters” box may not be ticked. In general the “Search for shortnearly exact matches” may be used in this respect, which provides mostof the above indicated settings. Further information in this respect maybe found in the “BLAST Program Selection Guide” published on the NCBIwebpage.

BLAST protein searches are performed with the BLASTp program. For thegeneral parameters, the “Max Target Sequences” box may be set to 100,the “Short queries” box may be ticked, the “Expect threshold” box may beset to 10 and the “Word Size” box may be set to “3”. For the scoringparameters the “Matrix” box may be set to “BLOSUM62”, the “Gap Costs”Box may be set to “Existence: 11 Extension: 1”, the “Compositionaladjustments” box may be set to “Conditional compositional score matrixadjustment”. For the Filters and Masking parameters the “Low complexityregions” box may not be ticked, the “Mask for lookup table only” box maynot be ticked and the “Mask lower case letters” box may not be ticked.

Modifications of both programs, e.g., in respect of the length of thesearched sequences, are performed according to the recommendations inthe “BLAST Program Selection Guide” published in a HTML and a PDFversion on the NCBI webpage.

Polynucleotides:

The term “polynucleotide” is intended to encompass a singular nucleicacid as well as plural nucleic acids, and refers to an isolated nucleicacid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA(pDNA). A polynucleotide may comprise a conventional phosphodiester bondor a non-conventional bond (e.g., an amide bond, such as found inpeptide nucleic acids (PNA)). The term “nucleic acid” refers to any oneor more nucleic acid segments, e.g., DNA or RNA fragments, present in apolynucleotide. By “isolated” nucleic acid or polynucleotide is intendeda nucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encodingan antibody contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. Isolated RNA molecules include in vivo orin vitro RNA transcripts of polynucleotides of the present invention.Isolated polynucleotides or nucleic acids according to the presentinvention further include such molecules produced synthetically. Inaddition, polynucleotide or a nucleic acid may be or may include aregulatory element such as a promoter, ribosome binding site, or atranscription terminator.

As used herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, introns, and the like, are not part of a coding region. Twoor more coding regions of the present invention can be present in asingle polynucleotide construct, e.g., on a single vector, or inseparate polynucleotide constructs, e.g., on separate (different)vectors. Furthermore, any vector may contain a single coding region, ormay comprise two or more coding regions, e.g., a single vector mayseparately encode an immunoglobulin heavy chain variable region and animmunoglobulin light chain variable region. In addition, a vector,polynucleotide, or nucleic acid of the invention may encode heterologouscoding regions, either fused or unfused to a nucleic acid encoding abinding molecule, an antibody, or fragment, variant, or derivativethereof. Heterologous coding regions include without limitationspecialized elements or motifs, such as a secretory signal peptide or aheterologous functional domain.

In certain embodiments, the polynucleotide or nucleic acid is DNA. Inthe case of DNA, a polynucleotide comprising a nucleic acid whichencodes a polypeptide normally may include a promoter and/or othertranscription or translation control elements operable associated withone or more coding regions. An operable association is when a codingregion for a gene product, e.g., a polypeptide, is associated with oneor more regulatory sequences in such a way as to place expression of thegene product under the influence or control of the regulatorysequence(s). Two DNA fragments (such as a polypeptide coding region anda promoter associated therewith) are “operable associated” or “operablelinked” if induction of promoter function results in the transcriptionof mRNA encoding the desired gene product and if the nature of thelinkage between the two DNA fragments does not interfere with theability of the expression regulatory sequences to direct the expressionof the gene product or interfere with the ability of the DNA template tobe transcribed. Thus, a promoter region would be operable associatedwith a nucleic acid encoding a polypeptide if the promoter was capableof effecting transcription of that nucleic acid. The promoter may be acell-specific promoter that directs substantial transcription of the DNAonly in predetermined cells. Other transcription control elements,besides a promoter, for example enhancers, operators, repressors, andtranscription termination signals, can be operable associated with thepolynucleotide to direct cell-specific transcription. Suitable promotersand other transcription control regions are disclosed herein.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions which function in vertebrate cells, such as, but not limited to,promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit β-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

In other embodiments, a polynucleotide of the present invention is RNA,for example, in the form of messenger RNA (mRNA).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. According to the signalhypothesis, proteins secreted by mammalian cells have a signal peptideor secretory leader sequence which is cleaved from the mature proteinonce export of the growing protein chain across the rough endoplasmicreticulum has been initiated. Those of ordinary skill in the art areaware that polypeptides secreted by vertebrate cells generally have asignal peptide fused to the N-terminus of the polypeptide, which iscleaved from the complete or “full-length” polypeptide to produce asecreted or “mature” form of the polypeptide. In certain embodiments,the native signal peptide, e.g., an immuno globulin heavy chain or lightchain signal peptide is used, or a functional derivative of thatsequence that retains the ability to direct the secretion of thepolypeptide that is operable associated with it. Alternatively, aheterologous mammalian signal peptide, or a functional derivativethereof, may be used. For example, the wild-type leader sequence may besubstituted with the leader sequence of human tissue plasminogenactivator (TPA) or mouse β-glucuronidase.

A “binding molecule” as used in the context of the present inventionrelates primarily to antibodies, and fragments thereof, but may alsorefer to other non-antibody molecules that bind to TTR including but notlimited to hormones, receptors, ligands, major histocompatibilitycomplex (MHC) molecules, chaperones such as heat shock proteins (HSPs)as well as cell-cell adhesion molecules such as members of the cadherin,intergrin, C-type lectin and immunoglobulin (Ig) superfamilies. Thus,for the sake of clarity only and without restricting the scope of thepresent invention most of the following embodiments are discussed withrespect to antibodies and antibody-like molecules which represent thepreferred binding molecules for the development of therapeutic anddiagnostic agents.

Antibodies:

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. An antibody or immunoglobulin is a binding molecule whichcomprises at least the variable domain of a heavy chain, and normallycomprises at least the variable domains of a heavy chain and a lightchain. Basic immunoglobulin structures in vertebrate systems arerelatively well understood; see, e.g., Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

As will be discussed in more detail below, the term “immunoglobulin”comprises various broad classes of polypeptides that can bedistinguished biochemically. Those skilled in the art will appreciatethat heavy chains are classified as gamma, mu, alpha, delta, or epsilon,(γ, μ, α, δ, ε) with some subclasses among them (e.g., γ1-γ4). It is thenature of this chain that determines the “class” of the antibody as IgG,IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses(isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc. are wellcharacterized and are known to confer functional specialization.Modified versions of each of these classes and isotypes are readilydiscernible to the skilled artisan in view of the instant disclosureand, accordingly, are within the scope of the instant invention. Allimmunoglobulin classes are clearly within the scope of the presentinvention, the following discussion will generally be directed to theIgG class of immunoglobulin molecules. With regard to IgG, a standardimmunoglobulin molecule comprises two identical light chain polypeptidesof molecular weight approximately 23,000 Daltons, and two identicalheavy chain polypeptides of molecular weight 53,000-70,000. The fourchains are typically joined by disulfide bonds in a “Y” configurationwherein the light chains bracket the heavy chains starting at the mouthof the “Y” and continuing through the variable region.

Light chains are classified as either kappa or lambda (κ, λ). Each heavychain class may be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (V_(L)) and heavy (V_(H)) chain portionsdetermine antigen recognition and specificity. Conversely, the constantdomains ofthe light chain (CL) and the heavy chain (CH1, CH2 or CH3)confer important biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention the numbering of the constant region domains increases asthey become more distal from the antigen-binding site or amino-terminusof the antibody. The N-terminal portion is a variable region and at theC-terminal portion is a constant region; the CH3 and CL domains actuallycomprise the carboxy-terminus of the heavy and light chain,respectively.

As indicated above, the variable region allows the antibody toselectively recognize and specifically bind epitopes on antigens. Thatis, the VL domain and VH domain, or subset ofthe complementaritydetermining regions (CDRs), of an antibody combine to form the variableregion that defines a three dimensional antigen-binding site. Thisquaternary antibody structure forms the antigen-binding site present atthe end of each arm of the Y. More specifically, the antigen-bindingsite is defined by three CDRs on each of the VH and VL chains. Anyantibody or immunoglobulin fragment which contains sufficient structureto specifically bind to TTR is denoted herein interchangeably as a“binding fragment” or an “immunospecific fragment.”

In naturally occurring antibodies, an antibody comprises sixhypervariable regions, sometimes called “complementarity determiningregions” or “CDRs” present in each antigen-binding domain, which areshort, non-contiguous sequences of amino acids that are specificallypositioned to form the antigen-binding domain as the antibody assumesits three dimensional configuration in an aqueous environment. The“CDRs” are flanked by four relatively conserved “framework” regions or“FRs” which show less inter-molecular variability. The framework regionslargely adopt a β-sheet conformation and the CDRs form loops whichconnect, and in some cases form part of, the β-sheet structure. Thus,framework regions act to form a scaffold that provides for positioningthe CDRs in correct orientation by inter-chain, non-covalentinteractions. The antigen-binding domain formed by the positioned CDRsdefines a surface complementary to the epitope on the immunoreactiveantigen. This complementary surface promotes the non-covalent binding ofthe antibody to its cognate epitope. The amino acids comprising the CDRsand the framework regions, respectively, can be readily identified forany given heavy or light chain variable region by one of ordinary skillin the art, since they have been precisely defined; see, “Sequences ofProteins of Immunological Interest,” Kabat, E., et al., U.S. Departmentof Health and Human Services, (1983); and Chothia and Lesk, J. Mol.Biol., 196 (1987), 901-917.

In the case where there are two or more definitions of a term which isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al., U.S. Dept. of Health and Human Services,“Sequences of Proteins of Immunological Interest” (1983) and by Chothiaand Lesk, J. Mol. Biol., 196 (1987), 901-917, which are incorporatedherein by reference, where the definitions include overlapping orsubsets of amino acid residues when compared against each other.Nevertheless, application of either definition to refer to a CDR of anantibody or variants thereof is intended to be within the scope of theterm as defined and used herein. The appropriate amino acid residueswhich encompass the CDRs as defined by each of the above citedreferences are set forth below in Table I as a comparison. The exactresidue numbers which encompass a particular CDR will vary depending onthe sequence and size of the CDR. Those skilled in the art can routinelydetermine which residues comprise a particular hypervariable region orCDR of the human IgG subtype of antibody given the variable region aminoacid sequence of the antibody.

TABLE I CDR Definitions¹ Kabat Chothia VH CDR1 31-35 26-32 VH CDR2 50-6552-58 VH CDR3  95-102  95-102 VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VLCDR3 89-97 91-96 ¹Numbering of all CDR definitions in Table I isaccording to the numbering conventions set forth by Kabat et al. (seebelow).

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an antibody or antigen-binding fragment,variant, or derivative thereof of the present invention are according tothe Kabat numbering system, which however is theoretical and may notequally apply to every antibody of the present invention. For example,depending on the position of the first CDR the following CDRs might beshifted in either direction.

Antibodies or antigen-binding fragments, immunospecific fragments,variants, or derivatives thereof of the invention include, but are notlimited to, polyclonal, monoclonal, multispecific, human, humanized,primatized, murinized or chimeric antibodies, single chain antibodies,epitope-binding fragments, e.g., Fab, Fab′ and F(ab′)₂, Fd, Fvs,single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs(sdFv), fragments comprising either a V_(L) or V_(H) domain, fragmentsproduced by a Fab expression library, and anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies disclosedherein). ScFv molecules are known in the art and are described, e.g., inU.S. Pat. No. 5,892,019 Immunoglobulin or antibody molecules of theinvention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY),class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule.

In one embodiment, the antibody of the present invention is not IgM or aderivative thereof with a pentavalent structure. Particular, in specificapplications of the present invention, especially therapeutic use, IgMsare less useful than IgG and other bivalent antibodies or correspondingbinding molecules since IgMs due to their pentavalent structure and lackof affinity maturation often show unspecific cross-reactivities and verylow affinity.

In a particularly preferred embodiment, the antibody of the presentinvention is not a polyclonal antibody, i.e. it substantially consistsof one particular antibody species rather than being a mixture obtainedfrom a plasma immunoglobulin sample.

Antibody fragments, including single-chain antibodies, may comprise thevariable region(s) alone or in combination with the entirety or aportion of the following: hinge region, CH1, CH2, and CH3 domains. Alsoincluded in the invention are TTR binding fragments which comprise anycombination of variable region(s) with a hinge region, CH1, CH2, and CH3domains. Antibodies or immunospecific fragments thereof of the presentinvention may be from any animal origin including birds and mammals.Preferably, the antibodies are human, murine, donkey, rabbit, goat,guinea pig, camel, llama, horse, or chicken antibodies. In anotherembodiment, the variable region may be condricthoid in origin (e.g.,from sharks).

In one aspect, the antibody of the present invention is a humanmonoclonal antibody isolated from a human. Optionally, the frameworkregion of the human antibody is aligned and adopted in accordance withthe pertinent human germ line variable region sequences in the database;see, e.g., Vbase (http://vbase.mrc-cpe.cam.ac.uk/) hosted by the MRCCentre for Protein Engineering (Cambridge, UK). For example, amino acidsconsidered to potentially deviate from the true germ line sequence couldbe due to the PCR primer sequences incorporated during the cloningprocess.

Compared to artificially generated human-like antibodies such as singlechain antibody fragments (scFvs) from a phage displayed antibody libraryor xenogeneic mice the human monoclonal antibody of the presentinvention is characterized by (i) being obtained using the human immuneresponse rather than that of animal surrogates, i.e. the antibody hasbeen generated in response to natural TTR in its relevant conformationin the human body, (ii) having protected the individual or is at leastsignificant for the presence of TTR, and (iii) since the antibody is ofhuman origin the risks of cross-reactivity against self-antigens isminimized. Thus, in accordance with the present invention the terms“human monoclonal antibody”, “human monoclonal autoantibody”, “humanantibody” and the like are used to denote a TTR binding molecule whichis of human origin, i.e. which has been isolated from a human cell suchas a B cell or hybridoma thereof or the cDNA of which has been directlycloned from mRNA of a human cell, for example a human memory B cell. Ahuman antibody is still “human”, i.e. human-derived even if amino acidsubstitutions are made in the antibody, e.g., to improve bindingcharacteristics.

In one embodiment the human-derived antibodies of the present inventioncomprises heterologous regions compared to the natural occurringantibodies, e.g. amino acid substitutions in the framework region,constant region exogenously fused to the variable region, differentamino acids at the C- or N-terminal ends and the like.

Antibodies derived from human immunoglobulin libraries or from animalstransgenic for one or more human immunoglobulins and that do not expressendogenous immunoglobulins, as described infra and, for example in, U.S.Pat. No. 5,939,598 by Kucherlapati et al., are denoted human-likeantibodies in order distinguish them from truly human antibodies of thepresent invention.

For example, the paring of heavy and light chains of human-likeantibodies such as synthetic and semi-synthetic antibodies typicallyisolated from phage display do not necessarily reflect the originalparing as it occurred in the original human B cell. Accordingly Fab andscFv fragments obtained from recombinant expression libraries ascommonly used in the prior art can be considered as being artificialwith all possible associated effects on immunogenicity and stability.

In contrast, the present invention provides isolated affinity-maturedantibodies from selected human subjects, which are characterized bytheir therapeutic utility and their tolerance in man.

As used herein, the term “rodentized antibody” or “rodentizedimmunoglobulin” refers to an antibody comprising one or more CDRs from ahuman antibody of the present invention; and a human framework regionthat contains amino acid substitutions and/or deletions and/orinsertions that are based on a rodent antibody sequence. When referredto rodents, preferably sequences originating in mice and rats are used,wherein the antibodies comprising such sequences are referred to as“murinized” or “ratinized” respectively. The human immunoglobulinproviding the CDRs is called the “parent” or “acceptor” and the rodentantibody providing the framework changes is called the “donor”. Constantregions need not be present, but if they are, they are usuallysubstantially identical to the rodent antibody constant regions, i.e. atleast about 85% to 90%, preferably about 95% or more identical. Hence,in some embodiments, a full-length murinized human heavy or light chainimmunoglobulin contains a mouse constant region, human CDRs, and asubstantially human framework that has a number of “murinizing” aminoacid substitutions. Typically, a “murinized antibody” is an antibodycomprising a murinized variable light chain and/or a murinized variableheavy chain. For example, a murinized antibody would not encompass atypical chimeric antibody, e.g., because the entire variable region of achimeric antibody is non-mouse. A modified antibody that has been“murinized” by the process of “murinization” binds to the same antigenas the parent antibody that provides the CDRs and is usually lessimmunogenic in mice, as compared to the parent antibody. The aboveexplanations in respect of “murinized” antibodies apply analogously foroder “rodentized” antibodies, such as “ratinized antibodies”, whereinrat sequences are used instead of the murine.

As used herein, the term “heavy chain portion” includes amino acidsequences derived from an immunoglobulin heavy chain. A polypeptidecomprising a heavy chain portion comprises at least one of: a CH1domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain,a CH2 domain, a CH3 domain, or a variant or fragment thereof. Forexample, a binding polypeptide for use in the invention may comprise apolypeptide chain comprising a CH1 domain; a polypeptide chaincomprising a CH1 domain, at least a portion of a hinge domain, and a CH2domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; apolypeptide chain comprising a CH1 domain, at least a portion of a hingedomain, and a CH3 domain, or a polypeptide chain comprising a CH1domain, at least a portion of a hinge domain, a CH2 domain, and a CH3domain. In another embodiment, a polypeptide of the invention comprisesa polypeptide chain comprising a CH3 domain. Further, a bindingpolypeptide for use in the invention may lack at least a portion of aCH2 domain (e.g., all or part of a CH2 domain). As set forth above, itwill be understood by one of ordinary skill in the art that thesedomains (e.g., the heavy chain portions) may be modified such that theyvary in amino acid sequence from the naturally occurring immunoglobulinmolecule.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof disclosed herein, the heavy chain portions of onepolypeptide chain of a multimer are identical to those on a secondpolypeptide chain of the multimer. Alternatively, heavy chainportion-containing monomers ofthe invention are not identical. Forexample, each monomer may comprise a different target binding site,forming, for example, a bispecific antibody or diabody.

In another embodiment, the antibodies, or antigen-binding fragments,variants, or derivatives thereof disclosed herein are composed of asingle polypeptide chain such as scFvs and are to be expressedintracellularly (intrabodies) for potential in vivo therapeutic anddiagnostic applications.

The heavy chain portions of a binding polypeptide for use in thediagnostic and treatment methods disclosed herein may be derived fromdifferent immunoglobulin molecules. For example, a heavy chain portionof a polypeptide may comprise a CH1 domain derived from an IgG1 moleculeand a hinge region derived from an IgG3 molecule. In another example, aheavy chain portion can comprise a hinge region derived, in part, froman IgG1 molecule and, in part, from an IgG3 molecule. In anotherexample, a heavy chain portion can comprise a chimeric hinge derived, inpart, from an IgG1 molecule and, in part, from an IgG4 molecule.

As used herein, the term “light chain portion” includes amino acidsequences derived from an immunoglobulin light chain. Preferably, thelight chain portion comprises at least one of a V_(L) or CL domain.

The minimum size of a peptide or polypeptide epitope for an antibody isthought to be about four to five amino acids. Peptide or polypeptideepitopes preferably contain at least seven, more preferably at leastnine and most preferably between at least about 15 to about 30 aminoacids. Since a CDR can recognize an antigenic peptide or polypeptide inits tertiary form, the amino acids comprising an epitope need not becontiguous, and in some cases, may not even be on the same peptidechain. In the present invention, a peptide or polypeptide epitoperecognized by antibodies of the present invention contains a sequence ofat least 4, at least 5, at least 6, at least 7, more preferably at least8, at least 9, at least 10, at least 15, at least 20, at least 25, orbetween about 15 to about 30 contiguous or non-contiguous amino acids ofTTR.

By “specifically binding”, or “specifically recognizing”, usedinterchangeably herein, it is generally meant that a binding molecule,e.g., an antibody binds to an epitope via its antigen-binding domain,and that the binding entails some complementarity between theantigen-binding domain and the epitope. According to this definition, anantibody is said to “specifically bind” to an epitope when it binds tothat epitope, via its antigen-binding domain more readily than it wouldbind to a random, unrelated epitope. The term “specificity” is usedherein to qualify the relative affinity by which a certain antibodybinds to a certain epitope. For example, antibody “A” may be deemed tohave a higher specificity for a given epitope than antibody “B,” orantibody “A” may be said to bind to epitope “C” with a higherspecificity than it has for related epitope “D”.

Where present, the term “immunological binding characteristics,” orother binding characteristics of an antibody with an antigen, in all ofits grammatical forms, refers to the specificity, affinity,cross-reactivity, and other binding characteristics of an antibody.

By “preferentially binding”, it is meant that the binding molecule,e.g., antibody specifically binds to an epitope more readily than itwould bind to a related, similar, homologous, or analogous epitope.Thus, an antibody which “preferentially binds” to a given epitope wouldmore likely bind to that epitope than to a related epitope, even thoughsuch an antibody may cross-react with the related epitope.

By way of non-limiting example, a binding molecule, e.g., an antibodymay be considered to bind a first epitope preferentially if it bindssaid first epitope with a dissociation constant (K_(D)) that is lessthan the antibody's K_(D) for the second epitope. In anothernon-limiting example, an antibody may be considered to bind a firstantigen preferentially if it binds the first epitope with an affinitythat is at least one order of magnitude less than the antibody's K_(D)for the second epitope. In another non-limiting example, an antibody maybe considered to bind a first epitope preferentially if it binds thefirst epitope with an affinity that is at least two orders of magnitudeless than the antibody's K_(D) for the second epitope.

In another non-limiting example, a binding molecule, e.g., an antibodymay be considered to bind a first epitope preferentially if it binds thefirst epitope with an off rate (k(off)) that is less than the antibody'sk(off) for the second epitope. In another non-limiting example, anantibody may be considered to bind a first epitope preferentially if itbinds the first epitope with an affinity that is at least one order ofmagnitude less than the antibody's k(off) for the second epitope. Inanother non-limiting example, an antibody may be considered to bind afirst epitope preferentially if it binds the first epitope with anaffinity that is at least two orders of magnitude less than theantibody's k(off) for the second epitope.

A binding molecule, e.g., an antibody or antigen-binding fragment,variant, or derivative disclosed herein may be said to bind TTR or afragment, variant or specific conformation thereof with an off rate(k(off)) of less than or equal to 5×10⁻² sec⁻¹, 10⁻² sec⁻¹, 5×10⁻³ sec⁻¹or 10⁻³ sec⁻¹. More preferably, an antibody of the invention may be saidto bind TTR or a fragment, variant or specific conformation thereof withan off rate (k(off)) less than or equal to 5×10⁻⁴ sec⁻¹, 10⁻⁴ sec⁻¹,5×10⁻⁵ sec⁻¹, or 10⁻⁵ sec⁻¹ 5×10⁻⁶ sec⁻¹, 10⁻⁶ sec⁻¹, 5×10⁻⁷ sec⁻¹ or10⁻⁷ sec⁻¹.

A binding molecule, e.g., an antibody or antigen-binding fragment,variant, or derivative disclosed herein may be said to bind TTR or afragment, variant or specific conformation thereof with an on rate(k(on)) of greater than or equal to 10³ M⁻¹ sec⁻¹, 5×10³ M⁻¹ sec⁻¹, 10⁴M⁻¹ sec⁻¹ or 5×10⁴ M⁻¹ sec⁻¹. More preferably, an antibody of theinvention may be said to bind TTR or a fragment, variant or specificconformation thereof with an on rate (k(on)) greater than or equal to10⁵ M⁻¹ sec⁻¹, 5×10⁵ M⁻¹ sec⁻¹, 10⁶ M⁻¹ sec⁻¹, or 5×10⁶ M⁻¹ sec⁻¹ or 10⁷M⁻¹ sec⁻¹.

A binding molecule, e.g., an antibody is said to competitively inhibitbinding of a reference antibody to a given epitope if it preferentiallybinds to that epitope to the extent that it blocks, to some degree,binding of the reference antibody to the epitope. Competitive inhibitionmay be determined by any method known in the art, for example,competition ELISA assays. An antibody may be said to competitivelyinhibit binding of the reference antibody to a given epitope by at least90%, at least 80%, at least 70%, at least 60%, or at least 50%.

As used herein, the term “affinity” refers to a measure of the strengthof the binding of an individual epitope with the CDR of a bindingmolecule, e.g., an immunoglobulin molecule; see, e.g., Harlow et al.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,2nd ed. (1988) at pages 27-28. As used herein, the term “avidity” refersto the overall stability of the complex between a population ofimmunoglobulins and an antigen, that is, the functional combiningstrength of an immunoglobulin mixture with the antigen; see, e.g.,Harlow at pages 29-34. Avidity is related to both the affinity ofindividual immunoglobulin molecules in the population with specificepitopes, and also the valences of the immunoglobulins and the antigen.For example, the interaction between a bivalent monoclonal antibody andan antigen with a highly repeating epitope structure, such as a polymer,would be one of high avidity. The affinity or avidity of an antibody foran antigen can be determined experimentally using any suitable method;see, for example, Berzofsky et al., “Antibody-Antigen Interactions” InFundamental Immunology, Paul, W. E., Ed., Raven Press New York, N Y(1984), Kuby, Janis Immunology, W. H. Freeman and Company New York, N Y(1992), and methods described herein. General techniques for measuringthe affinity of an antibody for an antigen include ELISA, RIA, andsurface plasmon resonance. The measured affinity of a particularantibody-antigen interaction can vary if measured under differentconditions, e.g., salt concentration, pH. Thus, measurements of affinityand other antigen-binding parameters, e.g., K_(D), IC₅₀, are preferablymade with standardized solutions of antibody and antigen, and astandardized buffer.

Binding molecules, e.g., antibodies or antigen-binding fragments,variants or derivatives thereof of the invention may also be describedor specified in terms of their cross-reactivity. As used herein, theterm “cross-reactivity” refers to the ability of an antibody, specificfor one antigen, to react with a second antigen; a measure ofrelatedness between two different antigenic substances. Thus, anantibody is cross reactive if it binds to an epitope other than the onethat induced its formation. The cross reactive epitope generallycontains many of the same complementary structural features as theinducing epitope, and in some cases, may actually fit better than theoriginal.

For example, certain antibodies have some degree of cross-reactivity, inthat they bind related, but non-identical epitopes, e.g., epitopes withat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, and at least 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody may be said to have littleor no cross-reactivity if it does not bind epitopes with less than 95%,less than 90%, less than 85%, less than 80%, less than 75%, less than70%, less than 65%, less than 60%, less than 55%, and less than 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody may be deemed “highlyspecific” for a certain epitope, if it does not bind any other analog,ortholog, or homolog of that epitope.

Binding molecules, e.g., antibodies or antigen-binding fragments,variants or derivatives thereof of the invention may also be describedor specified in terms of their binding affinity to TTR and/or mutated,misfolded, misassembled and/or aggregated TTR species and/or fragmentsthereof Preferred binding affinities include those with a dissociationconstant or Kd less than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M,10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M,10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M,5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M,or 10⁻¹⁵ M.

In one embodiment, the antibody of the present invention has a Kd fordifferent TTR isoforms as illustrated for the exemplary antibodies inTable V below, i.e a Kd of >300 nM for wild-type native TTR, and/or a Kdof ≦15 nM, preferably ≦5 nM, and most preferably ≦2 nM for denaturatedTTR, and/or a Kd of ≦35 nM, preferably of ≦20 nM for native TTR-V30M,and/or a Kd of ≦150 nM, preferably of ≦5 nM, and most preferably ≦2 nMfor native TTR-L55P.

As previously indicated, the subunit structures and three dimensionalconfiguration of the constant regions of the various immunoglobulinclasses are well known. As used herein, the term “V_(H) domain” includesthe amino terminal variable domain of an immunoglobulin heavy chain andthe term “CH1 domain” includes the first (most amino terminal) constantregion domain of an immunoglobulin heavy chain. The CH1 domain isadjacent to the V_(H) domain and is amino terminal to the hinge regionof an immunoglobulin heavy chain molecule.

As used herein the term “CH2 domain” includes the portion of a heavychain molecule that extends, e.g., from about residue 244 to residue 360of an antibody using conventional numbering schemes (residues 244 to360, Kabat numbering system; and residues 231-340, EU numbering system;see Kabat E A et al. op. cit). The CH2 domain is unique in that it isnot closely paired with another domain. Rather, two N-linked branchedcarbohydrate chains are interposed between the two CH2 domains of anintact native IgG molecule. It is also well documented that the CH3domain extends from the CH2 domain to the C-terminal of the IgG moleculeand comprises approximately 108 residues.

As used herein, the term “hinge region” includes the portion of a heavychain molecule that joins the CH1 domain to the CH2 domain. This hingeregion comprises approximately 25 residues and is flexible, thusallowing the two N-terminal antigen-binding regions to moveindependently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains; see Roux et al., J.Immunol. 161 (1998), 4083-4090.

As used herein the term “disulfide bond” includes the covalent bondformed between two sulfur atoms. The amino acid cysteine comprises athiol group that can form a disulfide bond or bridge with a second thiolgroup. In most naturally occurring IgG molecules, the CH1 and CL regionsare linked by a disulfide bond and the two heavy chains are linked bytwo disulfide bonds at positions corresponding to 239 and 242 using theKabat numbering system (position 226 or 229, EU numbering system).

As used herein, the terms “linked”, “fused” or “fusion” are usedinterchangeably. These terms refer to the joining together of two moreelements or components, by whatever means including chemical conjugationor recombinant means. An “in-frame fusion” refers to the joining of twoor more polynucleotide open reading frames (ORFs) to form a continuouslonger ORF, in a manner that maintains the correct translational readingframe of the original ORFs. Thus, a recombinant fusion protein is asingle protein containing two or more segments that correspond topolypeptides encoded by the original ORFs (which segments are notnormally so joined in nature). Although the reading frame is thus madecontinuous throughout the fused segments, the segments may be physicallyor spatially separated by, for example, in-frame linker sequence. Forexample, polynucleotides encoding the CDRs of an immunoglobulin variableregion may be fused, in-frame, but be separated by a polynucleotideencoding at least one immunoglobulin framework region or additional CDRregions, as long as the “fused” CDRs are co-translated as part of acontinuous polypeptide.

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, an RNA or polypeptide. The processincludes any manifestation of the functional presence of the gene withinthe cell including, without limitation, gene knockdown as well as bothtransient expression and stable expression. It includes withoutlimitation transcription of the gene into messenger RNA (mRNA), transferRNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) orany other RNA product, and the translation of mRNA into polypeptide(s).If the final desired product is a biochemical, expression includes thecreation of that biochemical and any precursors. Expression of a geneproduces a “gene product.” As used herein, a gene product can be eithera nucleic acid, e.g., a messenger RNA produced by transcription of agene, or a polypeptide which is translated from a transcript. Geneproducts described herein further include nucleic acids with posttranscriptional modifications, e.g., polyadenylation, or polypeptideswith post translational modifications, e.g., methylation, glycosylation,the addition of lipids, association with other protein subunits,proteolytic cleavage, and the like.

As used herein, the term “sample” refers to any biological materialobtained from a subject or patient. In one aspect, a sample can compriseblood, peritoneal fluid, CSF, saliva or urine. In other aspects, asample can comprise whole blood, blood plasma, blood serum, B cellsenriched from blood samples, and cultured cells (e.g., B cells from asubject). A sample can also include a biopsy or tissue sample includingneural tissue. In still other aspects, a sample can comprise whole cellsand/or a lysate of the cells. Blood samples can be collected by methodsknown in the art. In one aspect, the pellet can be resuspended byvortexing at 4° C. in 200 μl buffer (20 mM Tris, pH. 7.5, 0.5% Nonidet,1 mM EDTA, 1 mM PMSF, 0.1 M NaCl, IX Sigma Protease Inhibitor, and IXSigma Phosphatase Inhibitors 1 and 2). The suspension can be kept on icefor 20 min with intermittent vortexing. After spinning at 15,000×g for 5min at about 4° C., aliquots of supernatant can be stored at about −70°C.

Diseases:

Unless stated otherwise, the terms “disorder” and “disease” are usedinterchangeably herein and comprise any undesired physiological changein a subject, an animal, an isolated organ, tissue or cell/cell culture.

Transthyretin (TTR) amyloidosis is a pathophysiological mechanism atplay in many different diseases which are characterized by abnormaldeposition of the TTR protein in various tissues as a result of astructural (i.e. conformational) change of the TTR protein. Themisfolded and misassembled TTR protein is toxic and occurs often as aconsequence of mutations in the TTR gene. Misfolded TTR toxicity leadsto local tissue damages, which upon accumulation over time can lead toorgan dysfunction and even organ failure. There are many types oftissues and organs that are susceptible to TTR amyloidosis, such as theperipheral and autonomic nervous system, the heart, leptomeninges, eyes,tendons, ligaments or kidneys. The broad range of tissues that can beaffected by TTR amyloidosis is a reason for the diversity of symptomsthe patients with TTR amyloidosis exhibit. In fact, patients with TTRamyloidosis are clinically categorized as suffering from differentdiseases, depending on the tissue or organ that is the most affected byTTR amyloidosis and the corresponding symptoms.

On this basis, TTR amyloidosis has been classified in a neuropathicform, wherein the peripheral and autonomic nervous system are primarilyaffected and patients exhibit mostly pain, paresthesia, muscularweakness and autonomic dysfunction. There is also a cardiac form of TTRamyloidosis, wherein the heart is primarily affected and patientsexhibit mostly orthostatic hypo- or hyper-tension, arrhythmia andcardiomegaly. These two forms are not mutually exclusive, and manypatients present with a combination of the two. When TTR amyloidosisaffect other tissues, this can lead to vitreous opacity, dry eyes orglaucoma, proteinurea, hyperthyroxinemia, carpal tunnel syndrome orpreeclampsia.

Therefore, in one embodiment of the present invention the antibodies ofthe present invention, binding molecules having substantially the samebinding specificities of any one thereof, the polynucleotides, thevectors, the cells and/or peptides of the present invention are used forpreparation of a pharmaceutical or diagnostic composition forprophylactic and/or therapeutic treatment of TTR amyloidosis diseases,for monitoring disease progression and/or treatment response, and forthe diagnosis of diseases associated with TTR amyloidosis comprisingFamilial Amyloid Polyneuropathy (FAP), Familial Amyloid Cardiomyopathy(FAC), Senile Systemic Amyloidosis (SSA), leptomeningeal/Central NervousSystem (CNS) amyloidosis including Alzheimer disease, ocularamyloidosis, renal amyloidosis, hyperthyroxinemia, carpal tunnelsyndrome, rotator cuff tears and lumbar spinal stenosis, andpreeclampsia.

Treatment:

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the development of cardiacdeficiency. Beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which themanifestation of the condition or disorder is to be prevented.

If not stated otherwise the term “drug,” “medicine,” or “medicament” areused interchangeably herein and shall include but are not limited to all(A) articles, medicines and preparations for internal or external use,and any substance or mixture of substances intended to be used fordiagnosis, cure, mitigation, treatment, or prevention of disease ofeither man or other animals; and (B) articles, medicines andpreparations (other than food) intended to affect the structure or anyfunction of the body of man or other animals; and (C) articles intendedfor use as a component of any article specified in clause (A) and (B).The term “drug,” “medicine,” or “medicament” shall include the completeformula of the preparation intended for use in either man or otheranimals containing one or more “agents,” “compounds”, “substances” or“(chemical) compositions” as and in some other context also otherpharmaceutically inactive excipients as fillers, disintegrants,lubricants, glidants, binders or ensuring easy transport,disintegration, disaggregation, dissolution and biological availabilityof the “drug,” “medicine,” or “medicament” at an intended targetlocation within the body of man or other animals, e.g., at the skin, inthe stomach or the intestine. The terms “agent,” “compound”, or“substance” are used interchangeably herein and shall include, in a moreparticular context, but are not limited to all pharmacologically activeagents, i.e. agents that induce a desired biological or pharmacologicaleffect or are investigated or tested for the capability of inducing sucha possible pharmacological effect by the methods of the presentinvention.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, e.g., a humanpatient, for whom diagnosis, prognosis, prevention, or therapy isdesired.

Pharmaceutical Carriers:

Pharmaceutically acceptable carriers and administration routes can betaken from corresponding literature known to the person skilled in theart. The pharmaceutical compositions of the present invention can beformulated according to methods well known in the art; see for exampleRemington: The Science and Practice of Pharmacy (2000) by the Universityof Sciences in Philadelphia, ISBN 0-683-306472, Vaccine Protocols 2ndEdition by Robinson et al., Humana Press, Totowa, N.J., USA, 2003;Banga, Therapeutic Peptides and Proteins: Formulation, Processing, andDelivery Systems. 2nd Edition by Taylor and Francis. (2006), ISBN:0-8493-1630-8. Examples of suitable pharmaceutical carriers are wellknown in the art and include phosphate buffered saline solutions, water,emulsions, such as oil/water emulsions, various types of wetting agents,sterile solutions etc. Compositions comprising such carriers can beformulated by well-known conventional methods. These pharmaceuticalcompositions can be administered to the subject at a suitable dose.Administration of the suitable compositions may be effected by differentways. Examples include administering a composition containing apharmaceutically acceptable carrier via oral, intranasal, rectal,topical, intraperitoneal, intravenous, intramuscular, subcutaneous,subdermal, transdermal, intrathecal, and intracranial methods. Aerosolformulations such as nasal spray formulations include purified aqueousor other solutions of the active agent with preservative agents andisotonic agents. Such formulations are preferably adjusted to a pH andisotonic state compatible with the nasal mucous membranes.Pharmaceutical compositions for oral administration, such as singledomain antibody molecules (e.g., “nanobodies™”) etc. are also envisagedin the present invention. Such oral formulations may be in tablet,capsule, powder, liquid or semi-solid form. A tablet may comprise asolid carrier, such as gelatin or an adjuvant. Formulations for rectalor vaginal administration may be presented as a suppository with asuitable carrier; see also O'Hagan et al., Nature Reviews, DrugDiscovery 2(9) (2003), 727-735. Further guidance regarding formulationsthat are suitable for various types of administration can be found inRemington's Pharmaceutical Sciences, Mace Publishing Company,Philadelphia, Pa., 17th ed. (1985) and corresponding updates. For abrief review of methods for drug delivery see Langer, Science 249(1990), 1527-1533.

II. Antibodies of the Present Invention

The present invention generally relates to human-derived anti-TTRantibodies and antigen-binding fragments thereof, which preferablydemonstrate the immunological binding characteristics and/or biologicalproperties as outlined for the antibodies illustrated in the Examples.In accordance with the present invention human monoclonal antibodiesspecific for TTR were cloned from a pool of healthy human subjects.However, in another embodiment of the present invention, the humanmonoclonal anti-TTR antibodies might also be cloned from patientsshowing symptoms of a disease and/or disorder associated with TTRamyloidosis

In the course of the experiments performed in accordance with thepresent invention, antibodies present in the conditioned media ofcultured human memory B cell were evaluated for their capacity to bindto TTR and to more than 10 other proteins including bovine serum albumin(BSA). Only the B-cell supernatants able to bind to the TTR protein butnot to any of the other proteins in the screen were selected for furtheranalysis, including determination of the antibody class and light chainsubclass. The selected B-cells were then processed for antibody cloning.

In brief, this consisted in the extraction of messenger RNAs from theselected B-cells, retro-transcription by RT-PCR, amplification of theantibody-coding regions by PCR, cloning into plasmid vectors andsequencing. Selected human antibodies were then produced by recombinantexpression in HEK293 or CHO cells and purification, and subsequentlycharacterized for their capacity to bind human TTR protein. Thecombination ofvarious tests, e.g. recombinant expression of theantibodies in HEK293 or CHO cells and the subsequent characterization oftheir binding specificities towards human TTR protein, and theirdistinctive binding to pathologically misfolded, misassembled and/oraggregated forms thereof confirmed that for the first time humanantibodies have been cloned that are highly specific for TTR anddistinctively recognize and selectively bind the pathologicallyaggregated forms of TTR protein, such as TTR fibrils. In some cases,mouse chimeric antibodies were also generated on the basis of thevariable domains of the human antibodies of the present invention. Thesemouse chimeric antibodies have shown equal binding affinity, specificityand selectivity to human TTR as the human antibodies as shown in FIGS. 6and 9 and in Examples 4 and 8.

Thus, the present invention generally relates to recombinanthuman-derived monoclonal anti-TTR antibody and binding fragments,derivatives and variants thereof. In one embodiment of the invention,the antibody is capable of binding human TTR.

In one embodiment, the present invention is directed to an anti-TTRantibody, or antigen-binding fragment, variant or derivatives thereof,where the antibody specifically binds to the same epitope of TTR as areference antibody selected from the group consisting of NI-301.59F1,NI-301.35G11, NI-301.37F1 and NI-301.12D3. Epitope mapping identified asequence within the human TTR including amino acids 61-EEEFVEGIY-69 (SEQID NO: 49) as the unique linear epitope recognized by antibodyNI-301.59F1 ofthis invention, a sequence within the human TTR includingamino acids 53-GELHGLTTEEE-63 (SEQ ID NO: 50) as the unique linearepitope recognized by antibody NI-301.35G11 of this invention, asequence within the human TTR including amino acids 41-WEPFA-45 (SEQ IDNO: 51) as the unique linear epitope recognized by antibody NI-301.37F1(see FIG. 10 and Example 9). Therefore, in one embodiment the antibodyof the present invention is provided, wherein the antibody specificallybinds a TTR epitope which comprises the amino acid sequence EEEFVEGIY(SEQ ID NO: 49), GELHGLTTEEE (SEQ ID NO: 50), or WEPFA (SEQ ID NO: 51).

In this context, as explained in Example 9 the binding epitopes of theexemplary antibodies NI-301.59F1, NI301.35G11, and NI-301.37F1 have beenanalyzed by using a panel of 29 sequential peptides 15 amino acid longand 11 amino acid overlap (i.e. first peptide TTRaa₁₋₁₅; second peptideTTRaa₅₋₁₉; etc.), wherein antibody NI-301.59F1 and 301.35G11 recognizetwo overlapping peptides, (15 and 16) and (13 and 14), respectively, andantibody NI 301.37F1 recognizes three overlapping peptides (9, 10 and11); see Example 9 and FIG. 10.

Thus, with respect to the amino acid sequence of the mature TTRpolypeptide and corresponding peptide mapping this means that antibodyNI-301.59F1 binding to the epitope EEEFVEGIY (SEQ ID NO: 49) is capableof recognizing peptides having the amino acid sequence GLTTEEEFVEGIYKV(SEQ ID NO: 85) and EEEFVEGIYKVEIDT (SEQ ID NO: 86).

Likewise, anti-TTR antibody NI-301.35G11 binding to epitope GELHGLTTEEE(SEQ ID NO: 50) is capable of recognizing peptides having the amino acidsequence TSESGELHGLTTEEE (SEQ ID NO: 87) and GELHGLTTEEEFVEG (SEQ ID NO:88).

Similarly, anti-TTR antibody NI-301.37F1 which binds to the epitopeWEPFA (SEQ ID NO: 51) is capable of recognizing peptides with the aminoacid sequences

FRKAADDT WEPFA SG, (SEQ ID NO: 89) ADDT WEPFA SGKTSE, (SEQ ID NO: 90)and WEPFA SGKTSESGEL. (SEQ ID NO: 91)

Thus, the subject antibodies of the present invention illustrated in theExamples are different from antibodies which recognize any of thementioned epitopes in context additional N- and/or C-terminal aminoacids only. Therefore, in a preferred embodiment of the presentinvention, specific binding of an anti-TTR antibody to a TTR epitopewhich comprises the amino acid sequence EEEFVEGIY (SEQ ID NO: 49),GELHGLTTEEE (SEQ ID NO: 50), or WEPFA (SEQ ID NO: 51) is determined withsequential peptides 15 amino acid long and 11 amino acid overlap inaccordance with Example 9 and FIGS. 10A to D.

In this context, extended epitope mapping performed in accordance withthe present invention and described in Example 9 using a panel of 151sequential peptides 15 amino acid long and 14 amino acid overlap,wherein for each peptide the amino-acid in position 10 was replaced byan alanine for non-alanine amino-acids, whereas alanines were replacedby glycine or proline revealed that antibody NI-301.59F1 binds epitopeEEFXEGIY (TTRaa₆₁₋₆₈) and antibody NI-301.35G11 binds ELXGLTXE(TTRaa₅₄₋₆₁) while no further sequence requirements have been determinedfor the epitope of antibody NI-301.37F1. Accordingly, in anotherembodiment determination whether a given antibody binds to the sameepitope as antibodies NI-301.59F1, NI301.35G11, and NI-301.37F1 isperformed according Example 9 and FIGS. 10E to H.

It goes without saying that epitope mapping and determination whether agiven antibody binds the same epitope as a subject antibody used inExample 9 and shown in FIG. 10 can also be applied to any other anti-TTRantibody of the present invention described in the Examples with thevariable region depicted in FIG. 1A-1T.

Accordingly, the present invention generally relates to any anti-TTRantibody and antibody-like molecule which binds to the same epitope asan antibody illustrated in the Examples and having at least the CDRsand/or variable heavy and light region as depicted in any one of FIGS.1A-1T.

In a further embodiment, the antibody specifically binds the amino acidsequence GELHGLTTEEE (SEQ ID NO: 50) but not GELHGPTTEEE, correspondingto the TTR-L55P mutant epitope, or the antibody specifically binds theamino acid sequence WEPFA (SEQ ID NO: 51) but not WGPFA, correspondingto the TTR-E42G mutant epitope.

Furthermore, without intending to be bound by initial experimentalobservations as demonstrated in the Examples 3 to 8 and shown in FIGS.2, 3, 4, 7, and 9, the human monoclonal NI-301.59F1, NI-301.35G11, andNI-301.37F1 anti-TTR antibodies of the present invention are preferablycharacterized in specifically binding to pathological misfolded,misassembled or aggregated TTR and not substantially recognizing TTR inthe physiological form. Hence, the present invention provides a set ofhuman anti-TTR antibodies with binding properties particularly usefulfor diagnostic and therapeutic purposes. Thus, in one embodiment thepresent invention provides antibodies which are capable of specificallybinding pathologically aggregated forms of TTR.

In one embodiment, the antibody of the present invention exhibits thebinding properties of the exemplary NI-301.59F1, NI-301.35G11, andNI-301.37F1 antibodies as described in the Examples. The anti-TTRantibody of the present invention preferentially recognizespathologically altered TTR, such as mutated, misfolded, misassembled oraggregated TTR species and fragments thereof rather than physiologicalTTR. Thus, in one embodiment, the antibody of the present invention doesnot substantially recognize physiological TTR species.

The term “does not substantially recognize” when used in the presentapplication to describe the binding affinity of a molecule of a groupcomprising an antibody, a fragment thereof or a binding molecule for aspecific target molecule, antigen and/or conformation of the targetmolecule and/or antigen means that the molecule of the aforementionedgroup binds said molecule, antigen and/or conformation with a bindingaffinity which is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold or 9-fold less than the binding affinity of the moleculeof the aforementioned group for binding another molecule, antigen and/orconformation. Very often the dissociation constant (KD) is used as ameasure of the binding affinity. Sometimes, it is the EC50 on a specificassay as for example an ELISA assay that is used as a measure ofthebinding affinity. Preferably the term “does not substantially recognize”when used in the present application means that the molecule of theaforementioned group binds said molecule, antigen and/or conformationwith a binding affinity which is at least or 10-fold, 20-fold, 50-fold,100-fold, 1000-fold or 10000-fold less than the binding affinity of saidmolecule of the aforementioned group for binding to another molecule,antigen and/or conformation.

In addition, or alternatively, the anti-TTR antibody of the presentinvention binds to disease causing misfolded, misassembled or aggregatedforms of human TTR. In this context, the binding affinities may be inthe range as shown for the exemplary NI-301.59F1, NI-301.35G11, andNI-301.37F1 antibodies in FIG. 2, respective FIG. 10, i.e. having halfmaximal effective concentrations (EC50) of about 1 pM to 500 nM,preferably an EC50 of about 50 pM to 100 nM, most preferably an EC50 ofabout 1 nM to 20 nM for human aggregated TTR and aggregated recombinantTTR as shown for NI-301.59F1 and NI-301.35G11, or an EC50 of about 100pM to 1 nM for human aggregated TTR and aggregated recombinant TTR asshown for NI-301.37F1.

In particular, the anti-TTR antibody, binding fragment or derivativethereof has a binding affinity corresponding to an EC50 value of ≦5 nMfor binding aggregated wild-type and/or an EC50 of ≦20 nM, preferably≦10 nM and most preferably ≦1 nM for binding aggregated V30M-TTR; seeExample 3 and FIG. 2.

Some antibodies are able to bind to a wide array of biomolecules, e.g.,proteins. As the skilled artisan will appreciate, the term specific isused herein to indicate that other biomolecules than TTR proteins orfragments thereof do not significantly bind to the antigen-bindingmolecule, e.g., one of the antibodies of the present invention.Preferably, the level of binding to a biomolecule other than TTR resultsin a binding affinity which is at most only 20% or less, 10% or less,only 5% or less, only 2% or less or only 1% or less (i.e. at least 5,10, 20, 50 or 100 fold lower, or anything beyond that) of the affinityto TTR, respectively; see e.g., FIG. 2.

In one embodiment the anti-TTR antibody of the present invention bindspreferentially to aggregated forms of TTR, misfolded TTR, misassembledTTR, and/or fragments, derivatives, fibrils and/or oligomers thereof. Inanother embodiment the anti-TTR antibody of the present inventionpreferentially binds to both native TTR and pathologically misfolded,misassembled, or aggregated forms of TTR.

As mentioned before, amorphous and amyloid TTR deposits can lead todifferent diseases depending on where in the body the misfolded,misassembled, and/or aggregated TTR species or fragments thereof occur.For example patients with Familial Amyloid Polyneuropathy (FAP) exhibitTTR deposits primarily in the small diameter nerve fibers, and thereforepresent primarily symptoms such as altered sensory perceptions andautonomic dysfunctions, including gastro-intestinal dysfunctions orimpotence; Patients with Familial Amyloid Cardiomyopathy (FAC) or SenileSystemic Amyloidosis (SSA) exhibit TTR deposits primarily in the heart,and therefore present symptoms such as cardiac insufficiency or cardiacarrhythmia; Patients with TTR deposits in kidneys may present renaldysfunctions and proteinurea.

Therefore, in one embodiment the antibody of the present invention isuseful for the treatment of Familial Amyloid Polyneuropathy (FAP),Familial Amyloid Cardiomyopathy (FAC), Senile Systemic Amyloidosis(SSA), systemic familial amyloidosis, leptomeningeal/Central NervousSystem (CNS) amyloidosis including Alzheimer disease, ocularamyloidosis, renal amyloidosis, hyperthyroxinemia, ligament amyloidosisincluding carpal tunnel syndrome, rotator cuff tears and lumbar spinalstenosis, and preeclampsia, and symptoms thereof.

The present invention is also drawn to an antibody, or antigen-bindingfragment, variant or derivatives thereof, where the antibody comprisesan antigen-binding domain identical to that of an antibody selected fromthe group consisting of NI-301.59F1, NI-301.35G11, NI-301.37F1,NI-301.2F5, NI-301.28B3, NI-301.119C12, NI-301.5D8, NI-301.9D5,NI-301.104F5, NI-301.21F10, NI-301.9G12, NI-301.12D3, NI-301.44E4,NI-301.18C4, NI-301.11A10, NI-301.3C9, NI-301.14D8, NI-301.9X4, andNI-301.14C3.

The present invention further exemplifies several binding molecules,e.g., antibodies and binding fragments thereof, which may becharacterized by comprising in their variable region, e.g., bindingdomain at least one complementarity determining region (CDR) of theV_(H) and/or V_(L) variable region comprising any one of the amino acidsequences depicted in FIG. 1. The corresponding nucleotide sequencesencoding the above-identified variable regions are set forth in Table IIbelow. Exemplary sets of CDRs of the above amino acid sequences ofthe VHand/or VL region are depicted in FIG. 1. However, as discussed in thefollowing the person skilled in the art is well aware of the fact thatin addition or alternatively CDRs may be used, which differ in theiramino acid sequence from those set forth in FIG. 1 by one, two, three oreven more amino acids in case of CDR2 and

CDR3. Therefore, in one embodiment the antibody of the present inventionor a TTR-binding fragment thereof is provided comprising in its variableregion at least one complementarity determining region (CDR) as depictedin FIG. 1 and/or one or more CDRs thereof comprising one or more aminoacid substitutions.

In one embodiment, the antibody of the present invention is any one ofthe antibodies comprising an amino acid sequence of the VH and/or VLregion as depicted in FIG. 1 or a V_(H) and/or V_(L) region thereofcomprising one or more amino acid substitutions. Preferably, theantibody of the present invention is characterized by the preservationof the cognate pairing of the heavy and light chain as was present inthe human B-cell.

In a further embodiment of the present invention the anti-TTR antibody,TTR-binding fragment, synthetic or biotechnological variant thereof canbe optimized to have appropriate binding affinity to the target andpharmacokinetic properties. Therefore, at least one amino acid in theCDR or variable region, which is prone to modifications selected fromthe group consisting of glycosylation, oxidation, deamination, peptidebond cleavage, iso-aspartate formation and/or unpaired cysteine issubstituted by a mutated amino acid that lack such alteration or whereinat least one carbohydrate moiety is deleted or added chemically orenzymatically to the antibody. Examples for amino acid optimization canbe found in e.g. international applications WO 2010/121140 and WO2012/049570. Additional modification optimizing the antibody propertiesare described in Gavel et al., Protein Engineering 3 (1990), 433-442 andHelenius et al., Annu. Rev. Biochem. 73 (2004), 1019-1049.

Alternatively, the antibody of the present invention is an antibody orantigen-binding fragment, derivative or variant thereof, which competesfor binding to TTR with at least one of the antibodies having the VHand/or VL region as depicted in any one of FIG. 1 A to T.

Experimental results provided in FIG. 2 and Example 3 suggest that someof the anti-TTR antibodies of the present invention preferentially bindto disease causing misfolded, misassembled or aggregated forms of humananti-TTR over the physiological forms of the proteins. In one embodimentthus, the antibody of the present invention preferentially recognizesmisfolded, misassembled and/or aggregated TTR and/or fragment and/orderivatives thereof over physiological TTR.

The antibody of the present invention may be human, in particular fortherapeutic applications. Alternatively, the antibody of the presentinvention is a rodent, rodentized or chimeric rodent-human antibody,preferably a murine, murinized or chimeric murine-human antibody or arat, ratinized or chimeric rat-human antibody which are particularlyuseful for diagnostic methods and studies in animals. In one embodimentthe antibody of the present invention is a chimeric rodent-human or arodentized antibody.

Furthermore, in one embodiment, the chimeric antibody of the presentinvention, i.e. comprising the variable domains of a human antibody,e.g. NI-301.35G11 and generic murine light and heavy constant domains,exhibits the binding properties of the exemplary NI-301.mur35G11 murinechimeric antibodies as described in the Examples. Further, the mousechimeric antibodies of the present invention bind with a high affinityto human TTR as described in Example 4 and 8. Preferably, the bindingaffinity of chimeric antibodies is similar to their human counterparts.

In one embodiment the antibody of the present invention is provided bycultures of single or oligoclonal B-cells that are cultured and thesupernatant of the culture, which contains antibodies produced by saidB-cells, is screened for presence and affinity of anti-TTR antibodiestherein. The screening process comprises screening for binding to nativemonomeric, fibrillar or non-fibrillar aggregates like oligomers of hTTRderived from a synthetic full-length hTTR peptide or e.g. purified fromhuman plasma or recombinant expression.

In addition or alternatively the screening process for presence andaffinity of anti-TTR antibodies may comprise the steps of a sensitivetissue amyloid plaque immunoreactivity (TAPIR) assay such as describedin international application WO 2004/095031, the disclosure content ofwhich is incorporated herein by reference. Furthermore or alternatively,screens on renal, heart sections for binding to anti-TTR such asdescribed in analogy in international application WO 2008/081008 forbrain and spinal cord sections may be performed.

As mentioned above, due to its generation upon a human immune responsethe human monoclonal antibody of the present invention will recognizeepitopes which are of particular pathological relevance and which mightnot be accessible or less immunogenic in case of immunization processesfor the generation of, for example, mouse monoclonal antibodies and invitro screening of phage display libraries, respectively. Accordingly,it is prudent to stipulate that the epitope of the human anti-TTRantibody of the present invention is unique and no other antibody whichis capable of binding to the epitope recognized by the human monoclonalantibody of the present invention exists; see also FIG. 10. A furtherindication for the uniqueness of the antibodies of the present inventionis the fact that, as indicated in Example 8, antibodies NI-301.59F1,NI-301.35G11, and NI-301.37F1 of the present invention bind epitopesthat are specific for the misfolded, misassembled, and/or aggregated TTRconformation, which as indicated above, are of particular pathologicalrelevance and may not be obtainable by the usual processes for antibodygeneration, such as immunization or in vitro library screenings.

Therefore, in one embodiment the present invention also extendsgenerally to anti-TTR antibodies and TTR-binding molecules which competewith the human monoclonal antibody of the present invention for specificbinding to TTR. The present invention is more specifically directed toan antibody, or antigen-binding fragment, variant or derivativesthereof, where the antibody specifically binds to the same epitope ofTTR as a reference antibody selected from the group consisting ofNI-301.59F1, NI-301.35G11, NI-301.37F1 and/or NI-301.12D3.

Furthermore, in one embodiment the present invention also extendsgenerally to anti-TTR antibodies and TTR-binding molecules which competewith the human monoclonal antibody of the present invention for specificbinding to misfolded, misassembled and/or aggregated TTR species orfragments thereof. The present invention is therefore, more specificallyalso directed to an antibody, or antigen-binding fragment, variant orderivatives thereof, where the antibody specifically binds to the sameepitope of mis folded, misassembled or aggregated TTR species orfragments thereof as a reference antibody selected from the groupconsisting of NI-301.59F1, NI-301.35G11, NI-301.37F1 and/or NI-301.12D3.

Competition between antibodies is determined by an assay in which theimmunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen, such as TTR. Numerous types of competitivebinding assays are known, for example: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay; see Stahli et al.,Methods in Enzymology 9 (1983), 242-253; solid phase directbiotin-avidin EIA; see Kirkland et al., J. Immunol. 137 (1986),3614-3619 and Cheung et al., Virology 176 (1990), 546-552; solid phasedirect labeled assay, solid phase direct labeled sandwich assay; seeHarlow and Lane, Antibodies, A Laboratory Manual, Cold Spring HarborPress (1988); solid phase direct label RIA using I¹²⁵ label; see Morelet al., Molec. Immunol. 25 (1988), 7-15 and Moldenhauer et al., Scand.J. Immunol. 32 (1990), 77-82. Typically, such an assay involves the useof purified TTR or misfolded, misassembled or aggregated TTR, such asoligomers and/or fibrils thereof bound to a solid surface or cellsbearing either of these, an unlabeled test immunoglobulin and a labeledreference immunoglobulin, i.e. the human monoclonal antibody of thepresent invention. Competitive inhibition is measured by determining theamount of label bound to the solid surface or cells in the presence ofthe test immunoglobulin. Usually the test immunoglobulin is present inexcess. Preferably, the competitive binding assay is performed underconditions as described for the ELISA assay in the appended Examples.Antibodies identified by competition assay (competing antibodies)include antibodies binding to the same epitope as the reference antibodyand antibodies binding to an adjacent epitope sufficiently proximal tothe epitope bound by the reference antibody for steric hindrance tooccur. Usually, when a competing antibody is present in excess, it willinhibit specific binding of a reference antibody to a common antigen byat least 50% or 75%. Hence, the present invention is further drawn to anantibody, or antigen-binding fragment, variant or derivatives thereof,where the antibody competitively inhibits a reference antibody selectedfrom the group consisting of NI-301.59F1, NI-301.35G11, NI-301.37F1,NI-305.2F5, NI-301.28B3, NI-301.119C12, NI-301.5D8, NI-301.9D5,NI-301.104F5, NI-301.21F10, NI-301.9G12, NI-301.12D3, NI.301.44E4,NI-301.18C4 NI-301.11A10, NI-301.3C9, NI-301.14D8, NI-301.9X4, and/orNI-301.14C3 from binding to TTR.

In addition, the present invention is further drawn to an antibody, orantigen-binding fragment, variant or derivatives thereof, where theantibody competitively inhibits a reference antibody selected from thegroup consisting of NI-301.59F1, NI-301.35G11, NI-301.37F1, NI-305.2F5,NI-301.28B3, NI-301.119C12, NI-301.5D8, NI-301.9D5, NI-301.104F5,NI-301.21F10, NI-301.9G12, NI-301.12D3, NI-301.44E4 NI-301.18C4,NI-301.11A10, NI-301.3C9, NI-301.14D8, NI-301.9X4, and/or NI-301.14C3from binding to misfolded, misassembled or aggregated TTR species orfragments thereof.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (V_(H)), where at least oneof V_(H)-CDRs of the heavy chain variable region or at least two of theV_(H)-CDRs of the heavy chain variable region are at least 80%, 85%, 90%or 95% identical to reference heavy chain V_(H)-CDR1, V_(H)-CDR2 orV_(H)-CDR3 amino acid sequences from the antibodies disclosed herein.Alternatively, the V_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 regions of theV_(H) are at least 80%, 85%, 90% or 95% identical to reference heavychain V_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 amino acid sequences fromthe antibodies disclosed herein. Thus, according to this embodiment aheavy chain variable region of the invention has V_(H)-CDR1, V_(H)-CDR2and V_(H)-CDR3 polypeptide sequences related to the groups shown in FIG.1 respectively. While FIG. 1 shows V_(H)-CDRs defined by the Kabatsystem, other CDR definitions, e.g., V_(H)-CDRs defined by the Chothiasystem, are also included in the present invention, and can be easilyidentified by a person of ordinary skill in the art using the datapresented in FIG. 1.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (V_(H)) in which theV_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 regions have polypeptide sequenceswhich are identical to the V_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 groupsshown in FIG. 1 respectively.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (V_(H)) in which theV_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 regions have polypeptide sequenceswhich are identical to the V_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 groupsshown in FIG. 1 respectively, except for one, two, three, four, five, orsix amino acid substitutions in any one V_(H)-CDR. In certainembodiments the amino acid substitutions are conservative.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin light chain variable region (V_(L)), where at least oneof the V_(L)-CDRs of the light chain variable region or at least two ofthe V_(L)-CDRs of the light chain variable region are at least 80%, 85%,90% or 95% identical to reference light chain V_(L)-CDR1, V_(L)-CDR2 orV_(L)-CDR3 amino acid sequences from antibodies disclosed herein.Alternatively, the V_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 regions of theV_(L) are at least 80%, 85%, 90% or 95% identical to reference lightchain V_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 amino acid sequences fromantibodies disclosed herein. Thus, according to this embodiment a lightchain variable region of the invention has V_(L)-CDR1, V_(L)-CDR2 andV_(L)-CDR3 polypeptide sequences related to the polypeptides shown inFIG. 1 respectively. While FIG. 1 shows V_(L)-CDRs defined by the Kabatsystem, other CDR definitions, e.g., V_(L)-CDRs defined by the Chothiasystem, are also included in the present invention.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin light chain variable region (V_(L)) in which theV_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 regions have polypeptide sequenceswhich are identical to the V_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 groupsshown in FIG. 1 respectively.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (V_(L)) in which theV_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 regions have polypeptide sequenceswhich are identical to the V_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 groupsshown in FIG. 1 respectively, except for one, two, three, four, five, orsix amino acid substitutions in any one V_(L)-CDR. In certainembodiments the amino acid substitutions are conservative.

An immunoglobulin or its encoding cDNA may be further modified. Thus, ina further embodiment the method of the present invention comprises anyone of the step(s) of producing a chimeric antibody, murinized antibody,single-chain antibody, Fab-fragment, bi-specific antibody, fusionantibody, labeled antibody or an analog of any one of those.Corresponding methods are known to the person skilled in the art and aredescribed, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual”,CSH Press, Cold Spring Harbor (1988). When derivatives of saidantibodies are obtained by the phage display technique, surface plasmonresonance as employed in the BlAcore system can be used to increase theefficiency of phage antibodies which bind to the same epitope as that ofany one of the antibodies described herein (Schier, Human AntibodiesHybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995),7-13). The production of chimeric antibodies is described, for example,in international application WO 89/09622. Methods for the production ofhumanized antibodies are described in, e.g., European application EP-Al0 239 400 and international application WO 90/07861. Further sources ofantibodies to be utilized in accordance with the present invention areso-called xenogeneic antibodies. The general principle for theproduction of xenogeneic antibodies such as human-like antibodies inmice is described in, e.g., international applications WO 91/10741, WO94/02602, WO 96/34096 and WO 96/33735. As discussed above, the antibodyof the invention may exist in a variety of forms besides completeantibodies; including, for example, Fv, Fab and F(ab)₂, as well as insingle chains; see e.g. international application WO 88/09344. In oneembodiment therefore, the antibody of the present invention is provided,which is selected from the group consisting of a single chain Fvfragment (scFv), a F(ab′) fragment, a F(ab) fragment, and a F(ab′)₂fragment.

The antibodies of the present invention or their correspondingimmunoglobulin chain(s) can be further modified using conventionaltechniques known in the art, for example, by using amino aciddeletion(s), insertion(s), substitution(s), addition(s), and/orrecombination(s) and/or any other modification(s) known in the arteither alone or in combination. Methods for introducing suchmodifications in the DNA sequence underlying the amino acid sequence ofan immunoglobulin chain are well known to the person skilled in the art;see, e.g., Sambrook, Molecular Cloning A Laboratory Manual, Cold SpringHarbor Laboratory (1989) N.Y. and Ausubel, Current Protocols inMolecular Biology, Green Publishing Associates and Wiley Interscience,N.Y. (1994). Modifications of the antibody of the invention includechemical and/or enzymatic derivatizations at one or more constituentamino acids, including side chain modifications, backbone modifications,and N- and C-terminal modifications including acetylation,hydroxylation, methylation, amidation, and the attachment ofcarbohydrate or lipid moieties, cofactors, and the like. Likewise, thepresent invention encompasses the production of chimeric proteins whichcomprise the described antibody or some fragment thereof at the aminoterminus fused to heterologous molecule such as an immunostimulatoryligand at the carboxyl terminus; see, e.g., international application WO00/30680 for corresponding technical details.

Additionally, the present invention encompasses peptides including thosecontaining a binding molecule as described above, for example containingthe CDR3 region of the variable region of any one of the mentionedantibodies, in particular CDR3 of the heavy chain since it hasfrequently been observed that heavy chain CDR3 (HCDR3) is the regionhaving a greater degree of variability and a predominant participationin antigen-antibody interaction. Such peptides may easily be synthesizedor produced by recombinant means to produce a binding agent usefulaccording to the invention. Such methods are well known to those ofordinary skill in the art. Peptides can be synthesized for example,using automated peptide synthesizers which are commercially available.The peptides can also be produced by recombinant techniques byincorporating the DNA expressing the peptide into an expression vectorand transforming cells with the expression vector to produce thepeptide.

Hence, the present invention relates to any binding molecule, e.g., anantibody or binding fragment thereof which is oriented towards theanti-TTR antibodies and/or antibodies capable of binding mutated,misfolded, misassembled or aggregated TTR species and/or fragmentsthereof of the present invention and displays the mentioned properties,i.e. which specifically recognizes TTR and/or mutated, misfolded,misassembled or aggregated TTR species and/or fragments thereof. Suchantibodies and binding molecules can be tested for their bindingspecificity and affinity by ELISA and immunohistochemistry as describedherein, see, e.g., the Examples. These characteristics of the antibodiesand binding molecules can be tested by Western Blot as well.

The exemplary human antibody NI-301.37F1 showed prominent staining ofmisfolded TTR on sections from FAP patient skin biopsy but showed nostaining on healthy control pancreas, wherein pancreatic alpha cellsshow endogenous expression of TTR, i.e. native TTR (see Example 8 andFIG. 9). The exemplary antibodies NI-301.35G11 and NI-301.37F1 of thepresent invention also gave positive results on FAP mouse tissue showingabnormal TTR deposits in various tissues including the intestine; seeFIG. 8. This binding specificity towards pathological forms of TTR inhuman and animal tissue emphasizes besides the biochemical experimentsshowed herein (see FIG. 10) the usability of the antibodies of thepresent invention in treatment and diagnosis of diseases associated withTTR amyloidosis, which occurs preferably due to the occurrence ofmisfolded, misassembled, and/or aggregated TTR species and/or fragments,derivatives thereof

As an alternative to obtaining immunoglobulins directly from the cultureof B cells or memory B cells, the cells can be used as a source ofrearranged heavy chain and light chain loci for subsequent expressionand/or genetic manipulation. Rearranged antibody genes can be reversetranscribed from appropriate mRNAs to produce cDNA. If desired, theheavy chain constant region can be exchanged for that of a differentisotype or eliminated altogether. The variable regions can be linked toencode single chain Fv regions. Multiple Fv regions can be linked toconfer binding ability to more than one target or chimeric heavy andlight chain combinations can be employed. Once the genetic material isavailable, design of analogs as described above which retain both theirability to bind the desired target is straightforward. Methods for thecloning of antibody variable regions and generation of recombinantantibodies are known to the person skilled in the art and are described,for example, Gilliland et al., Tissue Antigens 47 (1996), 1-20; Doeneckeet al., Leukemia 11 (1997), 1787-1792.

Once the appropriate genetic material is obtained and, if desired,modified to encode an analog, the coding sequences, including those thatencode, at a minimum, the variable regions of the heavy and light chain,can be inserted into expression systems contained on vectors which canbe transfected into standard recombinant host cells. A variety of suchhost cells may be used; for efficient processing, however, mammaliancells are preferred. Typical mammalian cell lines useful for thispurpose include, but are not limited to, CHO cells, HEK 293 cells, orNSO cells.

The production of the antibody or analog is then undertaken by culturingthe modified recombinant host under culture conditions appropriate forthe growth of the host cells and the expression of the coding sequences.The antibodies are then recovered by isolating them from the culture.The expression systems are preferably designed to include signalpeptides so that the resulting antibodies are secreted into the medium;however, intracellular production is also possible.

In accordance with the above, the present invention also relates to apolynucleotide encoding the antibody or equivalent binding moleculeofthe present invention, in case of the antibody preferably at least avariable region of an immunoglobulin chain of the antibody describedabove. Typically, said variable region encoded by the polynucleotidecomprises at least one complementarity determining region (CDR) of theV_(H) and/or V_(L) of the variable region of the said antibody. In oneembodiment of the present invention, the polynucleotide is a cDNA.

The person skilled in the art will readily appreciate that the variabledomain of the antibody having the above-described variable domain can beused for the construction of other polypeptides or antibodies of desiredspecificity and biological function. Thus, the present invention alsoencompasses polypeptides and antibodies comprising at least one CDR ofthe above-described variable domain and which advantageously havesubstantially the same or similar binding properties as the antibodydescribed in the appended examples. The person skilled in the art knowsthat binding affinity may be enhanced by making amino acid substitutionswithin the CDRs or within the hypervariable loops (Chothia and Lesk, J.Mol. Biol. 196 (1987), 901-917) which partially overlap with the CDRs asdefined by Kabat; see, e.g., Riechmann, et al, Nature 332 (1988),323-327. Thus, the present invention also relates to antibodies whereinone or more of the mentioned CDRs comprise one or more, preferably notmore than two amino acid substitutions. Preferably, the antibody of theinvention comprises in one or both of its immunoglobulin chains two orall three CDRs of the variable regions as set forth in FIG. 1.

Binding molecules, e.g., antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention, as known by those ofordinary skill in the art, can comprise a constant region which mediatesone or more effector functions. For example, binding of the C1 componentof complement to an antibody constant region may activate the complementsystem. Activation of complement is important in the opsonization andlysis of cell pathogens. The activation of complement also stimulatesthe inflammatory response and may also be involved in autoimmunehypersensitivity. Further, antibodies bind to receptors on various cellsvia the Fc region, with a Fc receptor binding site on the antibody Fcregion binding to a Fc receptor (FcR) on a cell. There are a number ofFc receptors which are specific for different classes of antibody,including IgG (gamma receptors), IgE (epsilon receptors), IgA (alphareceptors) and IgM (mu receptors). Binding of antibody to Fc receptorson cell surfaces triggers a number of important and diverse biologicalresponses including engulfment and destruction of antibody-coatedparticles, clearance of immune complexes, lysis of antibody-coatedtarget cells by killer cells (called antibody-dependent cell-mediatedcytotoxicity, or ADCC), release of inflammatory mediators, placentaltransfer and control of immunoglobulin production.

Accordingly, certain embodiments of the present invention include anantibody, or antigen-binding fragment, variant, or derivative thereof,in which at least a fraction of one or more of the constant regiondomains has been deleted or otherwise altered so as to provide desiredbiochemical characteristics such as reduced effector functions, theability to non-covalently dimerize, increased ability to localize at thesite of TTR aggregation and deposition, reduced serum half-life, orincreased serum half-life when compared with a whole, unaltered antibodyof approximately the same immunogenicity. For example, certainantibodies for use in the diagnostic and treatment methods describedherein are domain deleted antibodies which comprise a polypeptide chainsimilar to an immunoglobulin heavy chain, but which lack at least aportion of one or more heavy chain domains. For instance, in certainantibodies, one entire domain of the constant region of the modifiedantibody will be deleted, for example, all or part of the CH2 domainwill be deleted. In other embodiments, certain antibodies for use in thediagnostic and treatment methods described herein have a constantregion, e.g., an IgG heavy chain constant region, which is altered toeliminate glycosylation, referred to elsewhere herein as aglycosylatedor “agly” antibodies. Such “agly” antibodies may be preparedenzymatically as well as by engineering the consensus glycosylationsite(s) in the constant region. While not being bound by theory, it isbelieved that “agly” antibodies may have an improved safety andstability profile in vivo. Methods of producing aglycosylatedantibodies, having desired effector function are found for example ininternational application WO 2005/018572, which is incorporated byreference in its entirety.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof described herein, the Fc portion may be mutated todecrease effector function using techniques known in the art. Forexample, the deletion or inactivation (through point mutations or othermeans) of a constant region domain may reduce Fc receptor binding of thecirculating modified antibody thereby increasing TTR localization. Inother cases it may be that constant region modifications consistent withthe instant invention moderate complement binding and thus reduce theserum half-life and nonspecific association of a conjugated cytotoxin.Yet other modifications of the constant region may be used to modifydisulfide linkages or oligosaccharide moieties that allow for enhancedlocalization due to increased antigen specificity or antibodyflexibility. The resulting physiological profile, bioavailability andother biochemical effects of the modifications, such as TTRlocalization, biodistribution and serum half-life, may easily bemeasured and quantified using well know immunological techniques withoutundue experimentation.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof described herein, the Fc portion may be mutated orexchanged for alternative protein sequences to increase the cellularuptake of antibodies by way of example by enhancing receptor-mediatedendocytosis of antibodies via Fcy receptors, LRP, or Thyl receptors orby ‘SuperAntibody Technology’, which is said to enable antibodies to beshuttled into living cells without harming them (Expert Opin. Biol.Ther. (2005), 237-241). For example, the generation of fusion proteinsof the antibody binding region and the cognate protein ligands of cellsurface receptors or bi- or multi-specific antibodies with a specificsequences binding to TTR as well as a cell surface receptor may beengineered using techniques known in the art.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof described herein, the Fc portion may be mutated orexchanged for alternative protein sequences or the antibody may bechemically modified to increase its blood brain barrier penetration.

Modified forms of antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can be made from whole precursor orparent antibodies using techniques known in the art. Exemplarytechniques are discussed in more detail herein. Antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention can be made or manufactured using techniques that are known inthe art. In certain embodiments, antibody molecules or fragments thereofare “recombinantly produced”, i.e., are produced using recombinant DNAtechnology. Exemplary techniques for making antibody molecules orfragments thereof are discussed in more detail elsewhere herein.

Antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention also include derivatives that are modified,e.g., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromspecifically binding to its cognate epitope. For example, but not by wayof limitation, the antibody derivatives include antibodies that havebeen modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

In particular preferred embodiments, antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention will notelicit a deleterious immune response in the animal to be treated, e.g.,in a human. In certain embodiments, binding molecules, e.g., antibodies,or antigen-binding fragments thereof of the invention are derived from apatient, e.g., a human patient, and are subsequently used in the samespecies from which they are derived, e.g., human, alleviating orminimizing the occurrence of deleterious immune responses.

De-immunization can also be used to decrease the immunogenicity of anantibody. As used herein, the term “de-immunization” includes alterationof an antibody to modify T cell epitopes; see, e.g., internationalapplications WO 98/52976 and WO 00/34317. For example, V_(H) and V_(L)sequences from the starting antibody are analyzed and a human T cellepitope “map” from each V region showing the location of epitopes inrelation to complementarity determining regions (CDRs) and other keyresidues within the sequence. Individual T cell epitopes from the T cellepitope map are analyzed in order to identify alternative amino acidsubstitutions with a low risk of altering activity of the finalantibody. A range of alternative VH and VL sequences are designedcomprising combinations of amino acid substitutions and these sequencesare subsequently incorporated into a range of binding polypeptides,e.g., TTR-specific antibodies or immunospecific fragments thereof foruse in the diagnostic and treatment methods disclosed herein, which arethen tested for function. Typically, between 12 and 24 variantantibodies are generated and tested. Complete heavy and light chaingenes comprising modified V and human C regions are then cloned intoexpression vectors and the subsequent plasmids introduced into celllines for the production of whole antibody. The antibodies are thencompared in appropriate biochemical and biological assays, and theoptimal variant is identified.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed.(1988); Hammerling et al., in: Monoclonal Antibodies and T-CellHybridomas Elsevier, N.Y., 563-681 (1981), said references incorporatedby reference in their entireties. The term “monoclonal antibody” as usedherein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced. Thus, the term“monoclonal antibody” is not limited to antibodies produced throughhybridoma technology. In certain embodiments, antibodies of the presentinvention are derived from human B cells which have been immortalizedvia transformation with Epstein-Barr virus, as described herein.

In the well-known hybridoma process (Kohler et al., Nature 256 (1975),495) the relatively short-lived, or mortal, lymphocytes from a mammal,e.g., B cells derived from a human subject as described herein, arefused with an immortal tumor cell line (e.g.,. a myeloma cell line),thus, producing hybrid cells or “hybridomas” which are both immortal andcapable of producing the genetically coded antibody of the B cell. Theresulting hybrids are segregated into single genetic strains byselection, dilution, and re-growth with each individual straincomprising specific genes for the formation of a single antibody. Theyproduce antibodies, which are homogeneous against a desired antigen and,in reference to their pure genetic parentage, are termed “monoclonal”.

Hybridoma cells thus prepared are seeded and grown in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, parental myeloma cells. Those skilledin the art will appreciate that reagents, cell lines and media for theformation, selection and growth of hybridomas are commercially availablefrom a number of sources and standardized protocols are wellestablished. Generally, culture medium in which the hybridoma cells aregrowing is assayed for production ofmonoclonal antibodies against thedesired antigen. The binding specificity of the monoclonal antibodiesproduced by hybridoma cells is determined by in vitro assays such asimmunoprecipitation, radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA) as described herein. After hybridoma cellsare identified that produce antibodies of the desired specificity,affinity and/or activity, the clones may be subcloned by limitingdilution procedures and grown by standard methods; see, e.g., Goding,Monoclonal Antibodies: Principles and Practice, Academic Press (1986),59-103. It will further be appreciated that the monoclonal antibodiessecreted by the subclones may be separated from culture medium, ascitesfluid or serum by conventional purification procedures such as, forexample, protein-A, hydroxylapatite chromatography, gel electrophoresis,dialysis or affinity chromatography.

In another embodiment, lymphocytes can be selected by micromanipulationand the variable genes isolated. For example, peripheral bloodmononuclear cells can be isolated from an immunized or naturally immunemammal, e.g., a human, and cultured for about 7 days in vitro. Thecultures can be screened for specific IgGs that meet the screeningcriteria. Cells from positive wells can be isolated. IndividualIg-producing B cells can be isolated by FACS or by identifying them in acomplement-mediated hemolytic plaque assay. Ig-producing B cells can bemicromanipulated into a tube and the V_(H) and V_(L) genes can beamplified using, e.g., RT-PCR. The V_(H) and V_(L) genes can be clonedinto an antibody expression vector and transfected into cells (e.g.,eukaryotic or prokaryotic cells) for expression.

Alternatively, antibody-producing cell lines may be selected andcultured using techniques well known to the skilled artisan. Suchtechniques are described in a variety of laboratory manuals and primarypublications. In this respect, techniques suitable for use in theinvention as described below are described in Current Protocols inImmunology, Coligan et al., Eds., Green Publishing Associates andWiley-Interscience, John Wiley and Sons, New York (1991) which is hereinincorporated by reference in its entirety, including supplements.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)₂ fragments may be producedrecombinantly or by proteolytic cleavage of immunoglobulin molecules,using enzymes such as papain (to produce Fab fragments) or pepsin (toproduce F(ab′)₂ fragments). F(ab′)₂ fragments contain the variableregion, the light chain constant region and the CH1 domain of the heavychain. Such fragments are sufficient for use, for example, inimmunodiagnostic procedures involving coupling the immunospecificportions of immunoglobulins to detecting reagents such as radioisotopes.

In one embodiment, an antibody of the invention comprises at least oneCDR of an antibody molecule. In another embodiment, an antibody of theinvention comprises at least two CDRs from one or more antibodymolecules. In another embodiment, an antibody of the invention comprisesat least three CDRs from one or more antibody molecules. In anotherembodiment, an antibody of the invention comprises at least four CDRsfrom one or more antibody molecules. In another embodiment, an antibodyof the invention comprises at least five CDRs from one or more antibodymolecules. In another embodiment, an antibody of the invention comprisesat least six CDRs from one or more antibody molecules. Exemplaryantibody molecules comprising at least one CDR that can be included inthe subject antibodies are described herein.

Antibodies of the present invention can be produced by any method knownin the art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably by recombinant expression techniques asdescribed herein.

In one embodiment, an antibody, or antigen-binding fragment, variant, orderivative thereof of the invention comprises a synthetic constantregion wherein one or more domains are partially or entirely deleted(“domain-deleted antibodies”). In certain embodiments compatiblemodified antibodies will comprise domain deleted constructs or variantswherein the entire CH2 domain has been removed (ΔCH2 constructs). Forother embodiments a short connecting peptide may be substituted for thedeleted domain to provide flexibility and freedom of movement for thevariable region. Those skilled in the art will appreciate that suchconstructs are particularly preferred due to the regulatory propertiesof the CH2 domain on the catabolic rate of the antibody. Domain deletedconstructs can be derived using a vector encoding an IgGi human constantdomain, see, e.g., international applications WO 02/060955 and WO02/096948A2. This vector is engineered to delete the CH2 domain andprovide a synthetic vector expressing a domain deleted IgGi constantregion.

In certain embodiments, antibodies, or antigen-binding fragments,variants, or derivatives thereof of the present invention areminibodies. Minibodies can be made using methods described in the art,see, e.g., U.S. Pat. No. 5,837,821 or international application WO94/09817.

In one embodiment, an antibody, or antigen-binding fragment, variant, orderivative thereof of the invention comprises an immunoglobulin heavychain having deletion or substitution of a few or even a single aminoacid as long as it permits association between the monomeric subunits.For example, the mutation of a single amino acid in selected areas ofthe CH2 domain may be enough to substantially reduce Fc binding andthereby increase TTR localization. Similarly, it may be desirable tosimply delete that part of one or more constant region domains thatcontrol the effector function (e.g. complement binding) to be modulated.Such partial deletions of the constant regions may improve selectedcharacteristics ofthe antibody (serum half-life) while leaving otherdesirable functions associated with the subject constant region domainintact. Moreover, as alluded to above, the constant regions of thedisclosed antibodies may be synthetic through the mutation orsubstitution of one or more amino acids that enhances the profile of theresulting construct. In this respect it may be possible to disrupt theactivity provided by a conserved binding site (e.g. Fc binding) whilesubstantially maintaining the configuration and immunogenic profile ofthe modified antibody. Yet other embodiments comprise the addition ofone or more amino acids to the constant region to enhance desirablecharacteristics such as an effector function or provide for morecytotoxin or carbohydrate attachment. In such embodiments it may bedesirable to insert or replicate specific sequences derived fromselected constant region domains.

The present invention also provides antibodies that comprise, consistessentially of, or consist of, variants (including derivatives) ofantibody molecules (e.g., the V_(H) regions and/or V_(L) regions)described herein, which antibodies or fragments thereofimmunospecifically bind to TTR. Standard techniques known to those ofskill in the art can be used to introduce mutations in the nucleotidesequence encoding an antibody, including, but not limited to,site-directed mutagenesis and PCR-mediated mutagenesis which result inamino acid substitutions. Preferably, the variants (includingderivatives) encode less than 50 amino acid substitutions, less than 40amino acid substitutions, less than 30 amino acid substitutions, lessthan 25 amino acid substitutions, less than 20 amino acid substitutions,less than 15 amino acid substitutions, less than 10 amino acidsubstitutions, less than 5 amino acid substitutions, less than 4 aminoacid substitutions, less than 3 amino acid substitutions, or less than 2amino acid substitutions relative to the reference V_(H) region,V_(H)-CDR1, V_(H)-CDR2, V_(H)-CDR3, V_(L) region, V_(L)-CDR1,V_(L)-CDR2, or V_(L)-CDR3. A “conservative amino acid substitution” isone in which the amino acid residue is replaced with an amino acidresidue having a side chain with a similar charge. Families of aminoacid residues having side chains with similar charges have been definedin the art. These families include amino acids with basic side chains(e.g., lysine, arginine, histidine), acidic side chains (e.g., asparticacid, glutamic acid), uncharged polar side chains (e.g., glycine,asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan), beta-branched side chains (e.g.,threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity (e.g., theability to bind TTR and/or misfolded, misassembled or aggregated TTRspecies and/or fragments thereof).

For example, it is possible to introduce mutations only in frameworkregions or only in CDR regions of an antibody molecule. Introducedmutations may be silent or neutral missense mutations, e.g., have no, orlittle, effect on an antibody's ability to bind antigen, indeed somesuch mutations do not alter the amino acid sequence whatsoever. Thesetypes of mutations may be useful to optimize codon usage, or improve ahybridoma's antibody production. Codon-optimized coding regions encodingantibodies of the present invention are disclosed elsewhere herein.Alternatively, non-neutral missense mutations may alter an antibody'sability to bind antigen. The location of most silent and neutralmissense mutations is likely to be in the framework regions, while thelocation of most non-neutral missense mutations is likely to be in CDR,though this is not an absolute requirement. One of skill in the artwould be able to design and test mutant molecules with desiredproperties such as no alteration in antigen-binding activity oralteration in binding activity (e.g., improvements in antigen-bindingactivity or change in antibody specificity). Following mutagenesis, theencoded protein may routinely be expressed and the functional and/orbiological activity of the encoded protein, (e.g., ability toimmunospecifically bind at least one epitope of TTR and/or mutated,misfolded, misassembled or aggregated TTR species and/or fragmentsthereof) can be determined using techniques described herein or byroutinely modifying techniques known in the art.

III. Polynucleotides Encoding Antibodies

A polynucleotide encoding an antibody, or antigen-binding fragment,variant, or derivative thereof can be composed of any polyribonucleotideor polydeoxribonucleotide, which may be unmodified

RNA or DNA or modified RNA or DNA. For example, a polynucleotideencoding an antibody, or antigen-binding fragment, variant, orderivative thereof can be composed of single- and double-stranded DNA,DNA that is a mixture of single- and double-stranded regions, single-and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or a mixtureof single-stranded and double-stranded regions. In addition, apolynucleotide encoding an antibody, or antigen-binding fragment,variant, or derivative thereof can be composed of triple-strandedregions comprising RNA or DNA or both RNA and DNA. A polynucleotideencoding an antibody, or antigen-binding fragment, variant, orderivative thereof may also contain one or more modified bases or DNA orRNA backbones modified for stability or for other reasons. “Modified”bases include, for example, tritylated bases and unusual bases such asinosine. A variety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

An isolated polynucleotide encoding a non-natural variant of apolypeptide derived from an immunoglobulin (e.g., an immunoglobulinheavy chain portion or light chain portion) can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of the immunoglobulin such that one or moreamino acid substitutions, additions or deletions are introduced into theencoded protein. Mutations may be introduced by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.Preferably, conservative amino acid substitutions are made at one ormore non-essential amino acid residues.

As is well known, RNA may be isolated from the original B cells,hybridoma cells or from other transformed cells by standard techniques,such as a guanidinium isothiocyanate extraction and precipitationfollowed by centrifugation or chromatography. Where desirable, mRNA maybe isolated from total RNA by standard techniques such as chromatographyon oligo dT cellulose. Suitable techniques are familiar in the art. Inone embodiment, cDNAs that encode the light and the heavy chains of theantibody may be made, either simultaneously or separately, using reversetranscriptase and DNA polymerase in accordance with well-known methods.PCR may be initiated by consensus constant region primers or by morespecific primers based on the published heavy and light chain DNA andamino acid sequences. As discussed above, PCR also may be used toisolate DNA clones encoding the antibody light and heavy chains. In thiscase the libraries may be screened by consensus primers or largerhomologous probes, such as human constant region probes.

DNA, typically plasmid DNA, may be isolated from the cells usingtechniques known in the art, restriction mapped and sequenced inaccordance with standard, well known techniques set forth in detail,e.g., in the foregoing references relating to recombinant DNAtechniques. Of course, the DNA may be synthetic according to the presentinvention at any point during the isolation process or subsequentanalysis.

In this context, the present invention also relates to a polynucleotideencoding at least the binding domain or variable region of animmunoglobulin chain of the antibody of the present invention. In oneembodiment, the present invention provides an isolated polynucleotidecomprising, consisting essentially of, or consisting of a nucleic acidencoding an immunoglobulin heavy chain variable region (V_(H)), where atleast one of the CDRs of the heavy chain variable region or at least twoof the V_(H)-CDRs of the heavy chain variable region are at least 80%,85%, 90%, or 95% identical to reference heavy chain V_(H)-CDR1,V_(H)-CDR2, or V_(H)-CDR3 amino acid sequences from the antibodiesdisclosed herein. Alternatively, the V_(H)-CDR1, V_(H)-CDR2, orV_(H)-CDR3 regions of the V_(H) are at least 80%, 85%, 90%, or 95%identical to reference heavy chain V_(H)-CDR1, V_(H)-CDR2, andV_(H)-CDR3 amino acid sequences from the antibodies disclosed herein.Thus, according to this embodiment a heavy chain variable region of theinvention has V_(H)-CDR1, V_(H)-CDR2, or V_(H)-CDR3 polypeptidesequences related to the polypeptide sequences shown in FIG. 1.

In another embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin light chain variable region(V_(L)), where at least one of the V_(L)-CDRs of the light chainvariable region or at least two of the V_(L)-CDRs of the light chainvariable region are at least 80%, 85%, 90%, or 95% identical toreference light chain V_(L)-CDR1, V_(L)-CDR2, or V_(L)-CDR3 amino acidsequences from the antibodies disclosed herein. Alternatively, theV_(L)-CDR1, V_(L)-CDR2, or V_(L)-CDR3 regions of the V_(L) are at least80%, 85%, 90%, or 95% identical to reference light chain V_(L)-CDR1,V_(L)-CDR2, and V_(L)-CDR3 amino acid sequences from the antibodiesdisclosed herein. Thus, according to this embodiment a light chainvariable region of the invention has V_(L)-CDR1, V_(L)-CDR2, orV_(L)-CDR3 polypeptide sequences related to the polypeptide sequencesshown in FIG. 1.

In another embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin heavy chain variable region(V_(H)) in which the V_(H)-CDR1, V_(H)-CDR2, and V_(H)-CDR3 regions havepolypeptide sequences which are identical to the V_(H)-CDR1, V_(H)-CDR2,and V_(H)-CDR3 groups shown in FIG. 1.

As known in the art, “sequence identity” between two polypeptides or twopolynucleotides is determined by comparing the amino acid or nucleicacid sequence of one polypeptide or polynucleotide to the sequence of asecond polypeptide or polynucleotide. When discussed herein, whether anyparticular polypeptide is at least about 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95% identical to another polypeptide can bedetermined using methods and computer programs/software known in the artsuch as, but not limited to, the BESTFIT program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).BESTFIT uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2 (1981), 482-489, to find the bestsegment of homology between two sequences. When using BESTFIT or anyother sequence alignment program to determine whether a particularsequence is, for example, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference polypeptide sequence and that gaps in homology of up to5% of the total number of amino acids in the reference sequence areallowed.

In a preferred embodiment of the present invention, the polynucleotidecomprises, consists essentially of, or consists of a nucleic acid havinga polynucleotide sequence of the V_(H) or V_(L) region of an anti-TTRantibody and/or antibody recognizing misfolded, misassembled oraggregated TTR species and/or fragments thereof as depicted in and TableII. In this respect, the person skilled in the art will readilyappreciate that the polynucleotides encoding at least the variabledomain of the light and/or heavy chain may encode the variable domain ofboth immunoglobulin chains or only one. In one embodiment therefore, thepolynucleotide comprises, consists essentially of, or consists of anucleic acid having a polynucleotide sequence of the V_(H) and the V_(L)region of an anti-TTR antibody recognizing misfolded, misassembled oraggregated TTR species and/or fragments thereof as depicted in Table II.

TABLE IINucleotide sequences of the V_(H) and V_(L) region of antibodiesrecognizing mutated, misfolded, misassembled or aggregatedTTR species and/or fragments thereof.Nucleotide sequences of variable heavy (VH) andvariable light (VL) chains or variable kappa- Antibody light chains (VK)NI-301.59F1-VH GAGGTGCAGCTGGTGGAGTCTGGGGGAGGGTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCACTTTTAGTAATTATTGGATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAATATAAATCAAGATAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCGCCATCTCCAGAGACAACTCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGGCGTGTATTACTGTGCGAGAGATCGCTATTGCAGTGGTGGGAGATGCTCCCGGGGTAACAACTGGTTCGACCCCTGGGGCCAGGGAACC CTGGTCACCGTCTCCTCGSEQ ID NO.: 1 NI-301.59F1-VLGAAATTGTGTTGACGCAGTCTCCAGCCACTCTGTCTCTGTCTCCAGGGGAGAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGAAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGATATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGTCTGAGGATTTTGCAGTTTATTACTGTCAGCAATATAATAACTGGCCTCCGTACACTTTTGGCCAGGGGACCAA AGTGGATATCAAASEQ ID NO.: 3 NI-301.35G11-VHGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCACTTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGTTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTAGTGGTAGTGGTGATACAACAAAATACACAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGGTGTTTCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCCCTATATTACTGTGTGAAAGATGGTAGTGGACGGATCGATCCTTTTGCTTTATGGGGCCAAGGGACAATGGTCACCGTCTCTTCG SEQ ID NO.: 5NI-301.35G11-VL GAAATTGTGATGACACAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGGTCTAGTCGTAGTCTCGTATACAGTGATGGAAACATTTACTTGAATTGGTTTCAGCAGAGGCCAGGCCAATCTCCAAGGCGCCTAATTTATAAGGTTTCTAACCGGGACTCTGGGGTCCCAGACAGATTCAGTGGCAGTGGGTCAGACACTGACTTCACACTGAGAATCAGCAGGGTGGAGGCTGAGGATGTTGGGGTCTATTACTGCATGCAGGGTACACACTGGCCTAGGACGTTCGGCCAAGGGACCAAGGTGGAGATCAAA SEQ ID NO.: 7 NI-301.37F1-VHCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCAGTGTCTCTGGTGGCTCCATCATCAGTAGGAGTTCCTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGGTATCTATCATAGTGGGAACACTTACGACAACCCGTCCCTCAAGAGTCGACTCACCATGTCCGTAGACACGTCGAAGAACCAGTTCTCCCTGAATCTGAGGTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGGATAGTGCCGGGGGGTGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCG SEQ ID NO.: 9NI-301.37F1-VL GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACAATCGCTTGCCGGGCCAGTCAGAGCGTTGGCACCTATTTAAATTGGTATCAGCAGAAAAGAGGGAAAGCCCCTAAACTCCTCATCTTTGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGACTTTGCAACTTACTACTGTCAACAGAGTTACAGTTCTCCTCCAACGTTCGGCCAAGGGACCAAGGT GGAGATCAAASEQ ID NO.: 11 NI-301.2F5-VHGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCGGTCTAGGAGGTCCCTGAGACTCTCCTGTGCAACCTCTGGATTCACCTTCAGTAACTATGCGATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCCATTATTTCATATGATGGAAACAATAAATACTACGCAGACTCCGTGAGGGGCCGATTCACCGTCTCCAGAGACAATTCCAAGAACACATTCTATCTGCAAATGAACAGCCTGAGAATTGAGGACACGGCTGTATATTTTTGTGCGAGAGGGAGCGGTAGAGCAGCTCGTCACTGGTTCGACCCCTGGGGCCAGGGCACCCTGGTCACCGTCTCC TCG SEQ ID NO.: 13NI-301.2F5-VL CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAATACCCAGGCAAAGCCCCCAAAGTCATGATTTTTGATGTTTTTAATCGGCCTTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGACTCCAGGCAGAGGACGAGGCTGATTATTACTGCAGTTCATATACAAGCAGCGTCACTCCTCACTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA SEQ ID NO.: 15 NI-301.28B3-VHCAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCCGGTGGCTCCATCACTAGTAGTAATTTCTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGCTATTTATTCTAGTGGAAACACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAAAAAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCTGACACGGCTGTCTATTACTGTGCGAGACACTCTTGTAGTAGTGCCAGCTGCTATCCTCCCGGTTTCTGGTTCGACCCCTGGGGCCAGGGAACC CTGGTCACCGTCTCCTCGSEQ ID NO.: 17 NI-301.28B3-VLGAAATTGTGATGACACAGTCTCCAGCCACCCTGTCTGCGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGACTGTTAGTTACAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCCGGCTCCTCATCTATGGCGCGTCCACCAGGGCCACTGGTATCCCAGGCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAATATAATAACTGGCCTCCGTGGACGTTCGGCCAAGGGACCA AGGTGGAAATCAAASEQ ID NO.: 19 NI-301.119C12-VHCAGGTGCAGCTGCAGGAGTCGGGCCCAAGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTGGTGTTTACTACTGGAGCTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGATATATTTCTAATACTGGGAACACCTACTACAACCCGTCCCTCAAGAGTCGAGTTACCATATCGATAGACACCTCCAAGAACCAGTTCTCCCTCAACCTGCGCTCTGTGACTGCCGCGGACACGGCCGACTATTTCTGTGCGAGAGAGTATTGTAGTGGTGGTAATTGCTACTCTCGCTTCTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCG SEQ ID NO.: 21 NI-301.119C12-VLCAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATCTCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGGTGTACACTGGTACCAGCAACTTTCAGGAACACCCCCCAAACTCCTCATCTATGGAGACAACAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGATGAGGCTCATTATTACTGCCAGTCCTATGACACCACCTTGAGTGGTTCGAGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA SEQ ID NO.: 23 NI-301.5D8-VHCAGGTGCAGCTACAGCAGTGGGGCGCAGGACGGTTGAAGCCTTCGGAGACCCTGTCCCTCACGTGCGCTGTCTATGGTGGGTCTTTCAGTGCTTACTACTGGAATTGGATCCGCCAGGCCCCAGGGAAGGGGCTGGAGTGGATTGGTGAAGTCAGTCATGGTGGCAGCAGCAACTACAGCCCGTCCCTCAGGGGTCGAGTCGCCATTTCTTTAGACACGTCCAAGAGCCAGTTCTCCCTGAGGCTGAATTCTGTGACCGCCGCGGACACGGCTGTTTATTACTGTGCGAGAGGCAGCCCTGTAGTACTACCAGGTGCCAGATTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO.: 25NI-301.5D8-VL CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTTTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGGAGTTATAACCTTGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCTTGATTTATGAGGTCAATAAGCGGCCCTCAGGAGTTTCTACTCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACGATCTCTGGGCTCCAGACTGAGGACGAGGCTGATTATTACTGCTGCTCATATGCAGGTAGTACTAAGGTCTTCGGAATTGGGAC CAAGGTCACCGTCCTASEQ ID NO.: 27 NI-301.9D5-VHCAGGTGCAGCTGCAGGAGTCGGGCCCAGGCCTGGTGAAGCCTTCAGAGACCCTGTCCCTCACCTGCATTGTCTCTGGTGTCTCCATCAGAAGTGGTGGTTACTACTGGAGCTGGATCCGGCAGCACCCAGGGAAGGGCCTGGAGTGGGTTGGGTTCATCTATTACACTGGGAACACCTACTACAACCCGTCCCTCAAGAGTCGAGCTACCATATCAGTAGACACCTCTAAGAACCAGTTCTCCCTGAGGCTGACCGCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGATTGTAGTGGTGGCAGCTGCCCCGAGTCCTACTTTGACTCCTGGGGTCGGGGCACCCTGGTCACC GTCTCCTCGSEQ ID NO.: 29 NI-301.9D5-VLGAAATTGTGATGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGTAGGGCCAGTCAGAGTGTTCGCAGTTTCTTAGCCTGGTACCAACAGAAATCTGGCCAGGCTCCCCGACTCCTCATCTATGATGCATCCAAGAGGGCCACTGGCATCCCAGCCAGGTTCAGTGACAGTGGGTCTGGAACAGACTTCACTCTCACCATCAGCAGACTAGAGACTGAAGACTCTGCGGTTTATTACTGTCAGCAGCGTACCAACTGGCCTCCACACCTCACTTTCGGCGGAGGGAC CAAGGTGGAAATCAAASEQ ID NO.: 31 NI-301.104F5-VHCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGAGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGGAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTTTGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCGTCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTCTACTACTGTGCAAGAGATGGTATAGCAGCCACTTATGCGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO.: 33NI-301.104F5-VL GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTCGCAGCTACTTAGCCTGGTACCAACAAAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTGGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAACGTAGCAACTGGCCGATCACCTTCGGCCAAGGGACACGAC TGGAGATTAAASEQ ID NO.: 35 NI-301.21F10-VHCAGGTGCAGCTGGTGGAGTCGGGGGGAGGTTTGGTCCAGCCTGGGGGGTCCTTGAGACTGTCCTGTGCGGTCTCTGGATTCACCCTTAGTAGTCTTAGTTCTTATTACATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCACTATAAACCCAGGTGGAAGTGAGAAGTCCTATGTGGACTCTGTGAAGGGCCGATTCACCGTCTCCAGAGACAACGCCAGGAGCTCAGTATATTTGCAAATGGACAGCCTGACAGTCGAGGACACGGCTATTTATTACTGTGCGAGACCAAGATATTGCACTAGTGGTGGTTGCTATTTTGACAACTGGGGCCAGGGAACCCTGG TCACCGTCTCCTCGSEQ ID NO.: 37 NI-301.21F10-VLCAGTCTGCCCTGACTCAGCCTCGCTCAGTGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGCAACCAATAGTGATGTTGGCGATTATAAGTCTGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCATGATTTATGATGTCGGTAGGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAATCTGACAACACGGCCTTCCTGACCATCTCTGGGCTCCAGACTGAGGATGAAGCTGATTACTTTTGCTGTATATATGTAGGCAGGTCTTCGGTGTTCGGCGGAGGGACC AAGTTGACCGTCCTGSEQ ID NO.: 39 NI-301.9G12-VHCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTCTGGTTTCTCCATCAGCAGTGGTTACTACTGGGGCTGGATCCGGCAGCCCCCAGGGACGGGGCTGGAGTGGATTGGGAGTATGTATCATAGTGGGAGGACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTGTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCCGTGTATTACTGTGCGAGGGGCTTCGATACTAGTGGTTCCCATCGGCCCCTCTCGACTGACTACTGGGGCCAGGGCACCCTGGTCACC GTCTCCTCGSEQ ID NO.: 41 NI-301.9G12-VLCAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAAGGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCCTGGTACCAGCAGCTCCCAGGAACAGCCCCCAAACTCCTCATTTATGACAATAATAAGCGACCCTCAGGGATTCCTGACCGAATCTCTGGCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACCGGACTCCAGACTGGGGACGAGGCCGATTATTACTGCGGAACCTGGGATAGCAGCCTGAGTGCTTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTA SEQ ID NO.: 43 NI-301.12D3-VHGAGGTGCAGCTGGTGGAGACTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGTAGCGTCTGGATTCACCTTCAGGAACTATGGCATGCACTGGGTCCGCCGGGCCCCAGGCAGGGGGCTGGAGTGGGTAGCAGTTATATGGTCTGATGGAAGTGATAAATACTATGCAGACTCCGTGGAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGGTGTTTCTCCAAATGAACAGCCTGAGAGCCGACGACACGGCTGTATACTTCTGTGCGAGAGAGCCGAGCAGCACCTGGGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO.: 45NI-301.12D3-VL CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGGGGTTATAACCTTGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGAGGACATTAAGGGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACAATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTTCTGCTGCTCATATGCAGGTACTGGCACTCTGGTATTCGGCGGAGG GACCAAGCTGACCGTCCTASEQ ID NO.: 47 NI-301.44E4-VHGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGATCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTAGTGGCAGTGGCAGTACGACATACTACGCAGACTCCGTGAAGGGCCGGTTCGCCATCTCCAGAGACAAATCCAAGAACACGCTGTCCCTACAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCAAAAGGGGCATGGGAGATACCCACCTACTTTGACAACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTC G SEQ ID NO.: 54NI-301.44E4-VK GAAATTGTGCTGACTCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTATTAGGAACAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCACTGGGTCTGGGACAGAGTTCACTCTCATCGTCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATAATAACTGGCCTCCCACGTGGACGTTCGGCCAAGGGA CCAAGGTGGAAATCAAASEQ ID NO.: 56 NI-301.18C4-VHGAGGTGCAGCTGGTGGAGTCTGGGGGAACCTTGGTCCAGCCGGGGGGGTCCCTGAGGCTCTCCTGCGCAGCGTCGGGATTCACATTCAACATTTATGCCATGACCTGGGTCCGCCTGTCTCCAGTGAGGGGACTGGAGTGGGTCTCTACTATTACTAGTGGTGGCGTCAGCATATATTACGCAGACTCCATAAAGGGCCGCTTCACCGTCTCCAGAGACAATGCCAAGAACATGGTGTTTCTACAACTGGACAACCTGACAGTCGATGACACGGCCATATATTACTGTGGGAAGGACGGAAACTGCGATGAGACAAGTTGTTACTTAAGGGGGATGGACGTCTGGGGCCAAGGGACCACG GTCACCGTCTCCTCGSEQ ID NO.: 61 NI-301.18C4-VLCAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCAGCGGCCCCAGGACAGAAGGTCACCATCTCCTGCTCTGGTAGCAGGTCCGACATTGGGTCTAAACTTGTTTCCTGGTACCAGGTAATCCCAGGAAGAGCCCCCCGGCTCGTCATTTTTGACACTTATAAGCGGCCCTCAGGGGTACCTGCCCGCTTCTCTGCCTCCAAGTCTGGCACGTCAGCCACCCTGGACATCGCCGGGCTCCAGCCTGGGGACGAGGCCGAATATTTCTGCGGATCATGGGGTAACAGTGAGAATTTTTATTATGTCTTCGGATCTGGGACCCGGGTCACCGTCCTG SEQ ID NO.: 63 NI-301.11A10-VHCAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTGTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATGCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATTTATTATAGTGGGAGCACCCTCTACAATCCGTCCCTCAAGAGTCGAGTCACCATGTCAATAGTCACGTCGAGGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCGGACACGGCCGTGTATTATTGTACCCGAATGGGGGAGGGGGGGCGGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO.: 65NI-301.11A10-VK GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAGTTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGGTCCTGATCTATGATGCCTCCAGTTTGGAAAGAGGGGTCCCATCAAGGTTCAGCGGCAGTGGGTCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTCTGCAACTTATTACTGCCAACACTATAATGGTTATTCAAGGACGTTCGGCCGCGGGACCAAG GTGGAAATCAAASEQ ID NO.: 67 NI-301.3C9 VHCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGTCTTCGCAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGCCTCCTTCACCAGGGGTGATTTCTACTGGAGTTGGATCCGCCAGGTCCCAGGGAAGGGCCTGGAATGGATTGGTTACATATATTCCACTGGGGACGTCTACTACAATCCGTCTCTCAAGAGTCGAGCAAACATCTCGGTCGACACGCCCAAGAAGCAGTTCTTCCTGAAATTGACCTCTTTGACTGCCGCAGACACGGCCGTCTATTTTTGTGCCAGGGAAGGACAATATTGTAGCGGTGGTAGTTGCTACCCTGAATACTGGGGCCAGGGAACCCTGGTCACC GTCTCCTCGSEQ ID NO.: 69 NI-301.3C9 VLTCCTATGAGCTGACTCAGCCACCCTCAGTGTCCGTGTCCCCAGGACAGACAGCCACCATCACCTGCTCTGGAGATAATTTGGGACATAAATTTACTTGCTGGTATCAGCAGAAGCCAGGCCAGTCCCCTGTCCTGGTCATCTATCAAGATCACAAGCGGCCCTCAGGGATCCCTGAGCGATTCTCCGGCTCCAACTCTGGGGACACAGCCACTCTGACCATCAGCGGGACCCAGGCTATGGATGAGGCTGAGTATTACTGTCAGGCGTGGGCCTTCCCCTATGTGGTCTTCGGCGGAGGGACCAAGCTGACC GTCCTASEQ ID NO.: 71 NI-301.14D8 VHGAGGTGCAGCTGGTGGAGACTGGGGGACGCTTGGTCCAGCCGGGGGGGTCCGTGAGACTCTCCTGTATAGCCTCTGGATTTCCCTTTAGGAATTATTGGATGAGTTGGGTCCGCCAGCCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAGGAAGATGGCAGTGACAGATACTATGTGGACTCTGTGAAGGGCCGCTTCACCATCTTTAGAGACAACGCCAAGAATTTTCTGAGTCTACAAATGAATCGCCTGAGAGCCGAGGACACGGCGGTATACTTCTGTGCGAGAATTGTAGGGGTAATCCCGTCCGCTGACCCATACTACCTTGACTCCTGGGGCCAGGGAACCCTGGTC ACCGTCTCCTCGSEQ ID NO.: 73 NI-301.14D8 VLCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTTTGCTGGACAGTCGGTCACCATCTCCTGCACTGGAACCAGCCTTAACATTGGGACTTACAACCTTATCTCCTGGTACCAACAACACCCAGGCAGAGCCCCCAGACTCATCATTTTTGAGGGCAATAGGCGGCCCCCCGGGATTTCTAATCGCTTCTCTGCCTCCAAGTCTGGCAACACGGCCTCCTTGACAGTCTCTGGGCTGCTGGCTGGCGACGAGGCTGATTATTACTGTTGCTCATTTGCAGGAAGAGTCTCTTTGGTGTTTGGCGGAGGG ACCAAGTTGACCGTCCTASEQ ID NO.: 75 NI-301.9X4 VHCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAACCTTCGGAGACCCTGTCCCTCACCTGCAGTGTCTCTGCTGGCTCCATCAGTAGTCACTACTGGAACTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAATGGATTGGGTCTATCTATCACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCACGTCTCCCTGAGGTTGACGTCTGTGACCGCCGCAGACACGGCCGTGTATTACTGTGCGAGAGACTACTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCG SEQ ID NO.: 77 NI-301.9X4 VLTCCTATGAGCTGACTCAGCCACCCTCGGTGTCAGTGTCCCCAGGACAGACGGCCAGGATCACCTGCTCTGGAGATGCGTTGCCAGACAAGTATGCTTATTGGTACCAGCAGAAGCCAGGCCAGGCCCCTATGTTGGTTATATATAAGGACAGTGAGAGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAGTTTGGGGACAACAGTCATGCTGACCATCAGTGGAGTCCAGGCAGAGGACGAGGCTGACTATTACTGTAAATCAGCAGACAGCAGTGGTACTTATTGGGTGTTCGGCGGGGGGACC AAGCTGACCGTCCTASEQ ID NO.: 79 NI-301.14C3 VHGAGGTGCAGCTGGTGGAGACTGGAGGAGGCTTGATCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGGTTCACCGTCAGTAGCCACTACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAATTATTTATAGCGGTGGTGGCACATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAGATCTACAGGTCGGGTAATACTGGTTATTCTTACGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCC TCG SEQ ID NO.: 81NI-301.14C3 VL TCCTATGAGCTGACTCAGCCACCCTCAGTGTCCGTGTCCCCAGGGCAGACAGCCAGCATCACCTGCTCTGGAGATAAATTGGGGAGTAAATATGCTTGCTGGTATCAGCAGAAGCCAGGCCAGTCCCCTGTACTGGTCATCTATGAAGATAAGAAGCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACAGCCACTCTGACCATCAGCGGGACCCAGGCTATGGATGAGGCTGACTATTTCTGTCAGGCGTGGGACAGCAGCACTTCTCATGTGGTATTCGGCGGAGGGACCA GGCTGACCGTCCTASEQ ID NO.: 83

Due to the cloning strategy the amino acid sequence at the N- andC-terminus of the heavy chain and light chains may potentially containprimer-induced alterations in FR1 and FR4, which however do notsubstantially affect the biological activity of the antibody. In orderto provide a consensus human antibody, the nucleotide and amino acidsequences of the original clone can be aligned with and tuned inaccordance with the pertinent human germ line variable region sequencesin the database; see, e.g., Vbase2, as described above. The amino acidsequence of human antibodies are indicated in bold when N- andC-terminus amino acids are considered to potentially deviate from theconsensus germ line sequence due to the PCR primer and thus have beenreplaced by primer-induced mutation correction (PIMC), see Table III.Accordingly, in one embodiment of the present invention, thepolynucleotide comprises, consists essentially of, or consists of anucleic acid having a polynucleotide sequence of the VH as depicted inTable III and the corresponding VL region of an anti-TTR antibody asshown in Table II.

TABLE IIINucleotide sequences of the V_(H) and V_(L) region of antibodiesrecognizing mutated, misfolded, misassembled or aggregatedTTR species and/or fragments thereof showing replacement by PIMC (bold).Alternative Antibody-regions with PIMCNucleotide sequences of variable heavy(V_(H)) chains NI-301.37F1-PIMC-CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACC VHCTGTCCCTCACCTGCAGTGTCTCTGGTGGCTCCATCATCAGTAGGAGTTCCTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGGTATCTATCATAGTGGGAACACTTACGACAACCCGTCCCTCAAGAGTCGACTCACCATGTCCGTAGACACGTCGAAGAACCAGTTCTCCCTGAATCTGAGGTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGGATAGTGCCGGGGGGTGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCG SEQ ID NO.: 52

The present invention also includes fragments of the polynucleotides ofthe invention, as described elsewhere. Additionally polynucleotideswhich encode fusion polynucleotides, Fab fragments, and otherderivatives, as described herein, are also contemplated by theinvention.

The polynucleotides may be produced or manufactured by any method knownin the art. For example, if the nucleotide sequence of the antibody isknown, a polynucleotide encoding the antibody may be assembled fromchemically synthesized oligonucleotides, e.g., as described in Kutmeieret al., BioTechniques 17 (1994), 242, which, briefly, involves thesynthesis of overlapping oligonucleotides containing portions of thesequence encoding the antibody, annealing and ligating of thoseoligonucleotides, and then amplification of the ligated oligonucleotidesby PCR.

Alternatively, a polynucleotide encoding an antibody, or antigen-bindingfragment, variant, or derivative thereof may be generated from nucleicacid from a suitable source. If a clone containing a nucleic acidencoding a particular antibody is not available, but the sequence of theantibody molecule is known, a nucleic acid encoding the antibody may bechemically synthesized or obtained from a suitable source (e.g., anantibody cDNA library, or a cDNA library generated from, or nucleicacid, preferably polyA⁺ RNA, isolated from, any tissue or cellsexpressing the TTR-specific antibody, such as hybridoma cells selectedto express an antibody) by PCR amplification using synthetic primershybridizable to the 3′ and 5′ ends of the sequence or by cloning usingan oligonucleotide probe specific for the particular gene sequence toidentify, e.g., a cDNA clone from a cDNA library that encodes theantibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.Accordingly, in one embodiment of the present invention the cDNAencoding an antibody, immunoglobulin chain, or fragment thereof is usedfor the production of an anti-TTR antibody.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody, or antigen-binding fragment, variant, or derivativethereof is determined, its nucleotide sequence may be manipulated usingmethods well known in the art for the manipulation of nucleotidesequences, e.g., recombinant DNA techniques, site directed mutagenesis,PCR, etc. (see, for example, the techniques described in Sambrook etal., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1990) and Ausubel et al., eds.,Current Protocols in Molecular Biology, John Wiley & Sons, NY (1998),which are both incorporated by reference herein in their entireties), togenerate antibodies having a different amino acid sequence, for exampleto create amino acid substitutions, deletions, and/or insertions.

IV. Expression of Antibody Polypeptides

Following manipulation of the isolated genetic material to provideantibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention, the polynucleotides encoding the antibodiesare typically inserted in an expression vector for introduction intohost cells that may be used to produce the desired quantity of antibody.Recombinant expression of an antibody, or fragment, derivative, oranalog thereof, e.g., a heavy or light chain of an antibody which bindsto a target molecule is described herein. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablelinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g.,international applications WO 86/05807 and WO 89/01036; and U.S. Pat.No. 5,122,464) and the variable domain of the antibody may be clonedinto such a vector for expression of the entire heavy or light chain.

The term “vector” or “expression vector” is used herein to mean vectorsused in accordance with the present invention as a vehicle forintroducing into and expressing a desired gene in a host cell. As knownto those skilled in the art, such vectors may easily be selected fromthe group consisting of plasmids, phages, viruses, and retroviruses. Ingeneral, vectors compatible with the instant invention will comprise aselection marker, appropriate restriction sites to facilitate cloning ofthe desired gene and the ability to enter and/or replicate in eukaryoticor prokaryotic cells. For the purposes of this invention, numerousexpression vector systems may be employed. For example, one class ofvector utilizes DNA elements which are derived from animal viruses suchas bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus,baculovirus, retroviruses (RSV, MMTV or MOMLV), or SV40 virus. Othersinvolve the use of polycistronic systems with internal ribosome bindingsites. Additionally, cells which have integrated the DNA into theirchromosomes may be selected by introducing one or more markers whichallow selection of transfected host cells. The marker may provide forprototrophy to an auxotrophic host, biocide resistance (e.g.,antibiotics), or resistance to heavy metals such as copper. Theselectable marker gene can either be directly linked to the DNAsequences to be expressed, or introduced into the same cell byco-transformation. Additional elements may also be needed for optimalsynthesis of mRNA. These elements may include signal sequences, splicesignals, as well as transcriptional promoters, enhancers, andtermination signals.

In particularly preferred embodiments the cloned variable region genesare inserted into an expression vector along with the heavy and lightchain constant region genes (preferably human) as discussed above. Thisvector contains the cytomegalovirus promoter/enhancer, the mouse betaglobin major promoter, the SV40 origin of replication, the bovine growthhormone polyadenylation sequence, neomycin phosphotransferase exon 1 andexon 2, the dihydrofolate reductase gene, and leader sequence. Thisvector has been found to result in very high level expression ofantibodies upon incorporation of variable and constant region genes,transfection in CHO cells, followed by selection in G418 containingmedium and methotrexate amplification. Of course, any expression vectorwhich is capable of eliciting expression in eukaryotic cells may be usedin the present invention. Examples of suitable vectors include, but arenot limited to plasmids pcDNA3, pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS,pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAX1, and pZeoSV2(available from Invitrogen, San Diego, Calif.), and plasmid pCI(available from Promega, Madison, Wis.). In general, screening largenumbers of transformed cells for those which express suitably highlevels if immunoglobulin heavy and light chains is routineexperimentation which can be carried out, for example, by roboticsystems. Vector systems are also taught in U.S. Pat. Nos. 5,736,137 and5,658,570, each of which is incorporated by reference in its entiretyherein. This system provides for high expression levels, e.g., >30pg/cell/day. Other exemplary vector systems are disclosed e.g., in U.S.Pat. No. 6,413,777.

In other preferred embodiments the antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention may beexpressed using polycistronic constructs such as those disclosed in USpatent application publication no. 2003-0157641 A1 and incorporatedherein in its entirety. In these expression systems, multiple geneproducts of interest such as heavy and light chains of antibodies may beproduced from a single polycistronic construct. These systemsadvantageously use an internal ribosome entry site (IRES) to providerelatively high levels of antibodies. Compatible IRES sequences aredisclosed in U.S. Pat. No. 6,193,980 which is also incorporated herein.Those skilled in the art will appreciate that such expression systemsmay be used to effectively produce the full range of antibodiesdisclosed in the instant application. Therefore, in one embodiment thepresent invention provides a vector comprising the polynucleotideencoding at least the binding domain or variable region of animmunoglobulin chain of the antibody, optionally in combination with apolynucleotide that encodes the variable region of the otherimmunoglobulin chain of said binding molecule.

More generally, once the vector or DNA sequence encoding a monomericsubunit of the antibody has been prepared, the expression vector may beintroduced into an appropriate host cell. Introduction of the plasmidinto the host cell can be accomplished by various techniques well knownto those of skill in the art. These include, but are not limited to,transfection including lipotransfection using, e.g., Fugene® orlipofectamine, protoplast fusion, calcium phosphate precipitation, cellfusion with enveloped DNA, microinjection, and infection with intactvirus. Typically, plasmid introduction into the host is via standardcalcium phosphate co-precipitation method. The host cells harboring theexpression construct are grown under conditions appropriate to theproduction of the light chains and heavy chains, and assayed for heavyand/or light chain protein synthesis. Exemplary assay techniques includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), orfluorescence-activated cell sorter analysis (FACS), immunohistochemistryand the like.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody for use in the methods describedherein. Thus, the invention includes host cells comprising apolynucleotide encoding an antibody of the invention, or a heavy orlight chain thereof, or at least the binding domain or variable regionof an immunoglobulin thereof, which preferably are operable linked to aheterologous promoter. In addition or alternatively the invention alsoincludes host cells comprising a vector, as defined hereinabove,comprising a polynucleotide encoding at least the binding domain orvariable region of an immunoglobulin chain of the antibody, optionallyin combination with a polynucleotide that encodes the variable region ofthe other immunoglobulin chain of said binding molecule. In preferredembodiments for the expression of double-chained antibodies, a singlevector or vectors encoding both the heavy and light chains may beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain is advantageouslyplaced before the heavy chain to avoid an excess of toxic free heavychain; see Proudfoot, Nature 322 (1986), 52; Kohler, Proc. Natl. Acad.Sci. USA 77 (1980), 2197. The coding sequences for the heavy and lightchains may comprise cDNA or genomic DNA.

As used herein, “host cells” refers to cells which harbor vectorsconstructed using recombinant DNA techniques and encoding at least oneheterologous gene. In descriptions of processes for isolation ofantibodies from recombinant hosts, the terms “cell” and “cell culture”are used interchangeably to denote the source of antibody unless it isclearly specified otherwise. In other words, recovery of polypeptidefrom the “cells” may mean either from spun down whole cells, or from thecell culture containing both the medium and the suspended cells.

A variety of host-expression vector systems may be utilized to expressantibody molecules for use in the methods described herein. Suchhost-expression systems represent vehicles by which the coding sequencesof interest may be produced and subsequently purified, but alsorepresent cells which may, when transformed or transfected with theappropriate nucleotide coding sequences, express an antibody molecule ofthe invention in situ. These include but are not limited tomicroorganisms such as bacteria (e.g., Escherichia coli, Bacillussubtilis) transformed with recombinant bacteriophage DNA, plasmid DNA orcosmid DNA expression vectors containing antibody coding sequences;yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeastexpression vectors containing antibody coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing antibody coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingantibody coding sequences; or mammalian cell systems (e.g., COS, CHO,NSO, BLK, 293, 3T3 cells) harboring recombinant expression constructscontaining promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably,bacterial cells such as E. coli, and more preferably, eukaryotic cells,especially for the expression of whole recombinant antibody molecule,are used for the expression of a recombinant antibody molecule. Forexample, mammalian cells such as Chinese Hamster Ovary (CHO) cells, inconjunction with a vector such as the major intermediate early genepromoter element from human cytomegalovirus is an effective expressionsystem for antibodies; see, e.g., Foecking et al., Gene 45 (1986), 101;Cockett et al., Bio/Technology 8 (1990), 2.

The host cell line used for protein expression is often of mammalianorigin; those skilled in the art are credited with ability topreferentially determine particular host cell lines which are bestsuited for the desired gene product to be expressed therein. Exemplaryhost cell lines include, but are not limited to, CHO (Chinese HamsterOvary), DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA(human cervical carcinoma), CVI (monkey kidney line), COS (a derivativeof CVI with SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK,WI38, R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast),HAK (hamster kidney line), SP2/O (mouse myeloma), P3x63-Ag3.653 (mousemyeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocyte)and 293 (human kidney). CHO and 293 cells are particularly preferred.Host cell lines are typically available from commercial services, theAmerican Tissue Culture Collection or from published literature.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which stably express theantibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11(1977), 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalskaand Szybalski, Proc. Natl. Acad. Sci. USA 48 (1992), 202), and adeninephosphoribosyltransferase (Lowy et al., Cell 22 (1980), 817) genes canbe employed in tk-, hgprt- or aprt-cells, respectively. Also,anti-metabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77 (1980), 357; O'Hare et al., Proc. Natl.Acad. Sci. USA 78 (1981), 1527); gpt, which confers resistance tomycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78(1981), 2072); neo, which confers resistance to the aminoglycoside G-418Goldspiel et al., Clinical Pharmacy 12 (1993), 488-505; Wu and Wu,Biotherapy 3 (1991), 87-95; Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32(1993), 573-596; Mulligan, Science 260 (1993), 926-932; and Morgan andAnderson, Ann. Rev. Biochem. 62 (1993), 191-217; TIB TECH 11 (1993),155-215; and hygro, which confers resistance to hygromycin (Santerre etal., Gene 30 (1984), 147. Methods commonly known in the art ofrecombinant DNA technology which can be used are described in Ausubel etal. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons,NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual,Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al.(eds), Current Protocols in Human Genetics, John Wiley & Sons, NY(1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification, for a review; see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Academic Press, New York, Vol. 3.(1987). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase; see Crouse et al., Mol. Cell. Biol. 3(1983), 257.

In vitro production allows scale-up to give large amounts of the desiredpolypeptides. Techniques for mammalian cell cultivation under tissueculture conditions are known in the art and include homogeneoussuspension culture, e.g. in an airlift reactor or in a continuousstirrer reactor, or immobilized or entrapped cell culture, e.g. inhollow fibers, microcapsules, on agarose microbeads or ceramiccartridges. If necessary and/or desired, the solutions of polypeptidescan be purified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose or (immuno-) affinity chromatography, e.g., afterpreferential biosynthesis of a synthetic hinge region polypeptide orprior to or subsequent to the HIC chromatography step described herein.

Genes encoding antibodies, or antigen-binding fragments, variants orderivatives thereof of the invention can also be expressed innon-mammalian cells such as bacteria or insect or yeast or plant cells.Bacteria which readily take up nucleic acids include members of theenterobacteriaceae, such as strains of E. coli or Salmonella;Bacillaceae, such as B. subtilis; Pneumococcus; Streptococcus, andHaemophilus influenzae. It will further be appreciated that, whenexpressed in bacteria, the heterologous polypeptides typically becomepart of inclusion bodies. The heterologous polypeptides must beisolated, purified and then assembled into functional molecules. Wheretetravalent forms of antibodies are desired, the subunits will thenself-assemble into tetravalent antibodies; see, e.g., internationalapplication WO 02/096948.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2 (1983),1791), in which the antibody coding sequence may be ligated individuallyinto the vector in frame with the lacZ coding region so that a fusionprotein is produced; pIN vectors (Inouye and Inouye, Nucleic Acids Res.13 (1985), 3101-3109; Van Heeke and Schuster, J. Biol. Chem. 24 (1989),5503-5509); and the like. pGEX vectors may also be used to expressforeign polypeptides as fusion proteins with glutathione S-transferase(GST). In general, such fusion proteins are soluble and can easily bepurified from lysed cells by adsorption and binding to a matrix ofglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene product can bereleased from the GST moiety.

In addition to prokaryotes, eukaryotic microbes may also be used.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among eukaryotic microorganisms although a number of other strainsare commonly available, e.g., Pichia pastoris. For expression inSaccharomyces, the plasmid YRp7, for example, (Stinchcomb et al., Nature282 (1979), 39; Kingsman et al., Gene 7 (1979), 141; Tschemper et al.,Gene 10 (1980), 157) is commonly used. This plasmid already contains theTRP 1 gene which provides a selection marker for a mutant strain ofyeast lacking the ability to grow in tryptophan, for example ATCC No.44076 or PEP4-1 (Jones, Genetics 85 (1977), 12). The presence of thetrpl lesion as a characteristic of the yeast host cell genome thenprovides an effective environment for detecting transformation by growthin the absence of tryptophan.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is typically used as a vector to express foreign genes. Thevirus grows in Spodoptera frugiperda cells. The antibody coding sequencemay be cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

Once an antibody molecule of the invention has been recombinantlyexpressed, the whole antibodies, their dimers, individual light andheavy chains, or other immunoglobulin forms of the present invention,can be purified according to standard procedures of the art, includingfor example, by chromatography (e.g., ion exchange, affinity,particularly by affinity for the specific antigen after Protein A, andsizing column chromatography), centrifugation, differential solubility,e.g. ammonium sulfate precipitation, or by any other standard techniquefor the purification of proteins; see, e.g., Scopes, “ProteinPurification”, Springer Verlag, N.Y. (1982). Alternatively, a preferredmethod for increasing the affinity of antibodies of the invention isdisclosed in US patent publication 2002-0123057 A1. In one embodimenttherefore, the present invention also provides a method for preparing ananti-TTR antibody or an antibody recognizing mutated, misfolded,misassembled or aggregated TTR species and/or fragments thereof orimmunoglobulin chain(s) thereof, said method comprising:

-   -   (a) culturing the host cell as defined hereinabove, which cell        comprised a polynucleotide or a vector as defined hereinbefore;        and    -   (b) isolating said antibody or immunoglobulin chain(s) thereof        from the culture.

Furthermore, in one embodiment the present invention also relates to anantibody or immunoglobulin chain(s) thereof encoded by a polynucleotideas defined hereinabove or obtainable by the method for preparing ananti-TTR antibody.

V. Fusion Proteins and Conjugates

In certain embodiments, the antibody polypeptide comprises an amino acidsequence or one or more moieties not normally associated with anantibody. Exemplary modifications are described in more detail below.For example, a single-chain Fv antibody fragment of the invention maycomprise a flexible linker sequence, or may be modified to add afunctional moiety (e.g., PEG, a drug, a toxin, or a label such as afluorescent, radioactive, enzyme, nuclear magnetic, heavy metal and thelike)

An antibody polypeptide of the invention may comprise, consistessentially of, or consist of a fusion protein. Fusion proteins arechimeric molecules which comprise, for example, an immunoglobulinTTR-binding domain with at least one target binding site, and at leastone heterologous portion, i.e., a portion with which it is not naturallylinked in nature. The amino acid sequences may normally exist inseparate proteins that are brought together in the fusion polypeptide orthey may normally exist in the same protein but are placed in a newarrangement in the fusion polypeptide. Fusion proteins may be created,for example, by chemical synthesis, or by creating and translating apolynucleotide in which the peptide regions are encoded in the desiredrelationship.

The term “heterologous” as applied to a polynucleotide or a polypeptide,means that the polynucleotide or polypeptide is derived from a distinctentity from that of the rest of the entity to which it is beingcompared. For instance, as used herein, a “heterologous polypeptide” tobe fused to an antibody, or an antigen-binding fragment, variant, oranalog thereof is derived from a non-immunoglobulin polypeptide of thesame species, or an immunoglobulin or non-immunoglobulin polypeptide ofa different species. As discussed in more detail elsewhere herein,antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalent and non-covalent conjugations) topolypeptides or other compositions. For example, antibodies may berecombinantly fused or conjugated to molecules useful as labels indetection assays and effector molecules such as heterologouspolypeptides, drugs, radionuclides, or toxins; see, e.g., internationalapplications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and European patent application EP 0 396 387.

Antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention can be composed of amino acids joined to eachother by peptide bonds or modified peptide bonds, i.e., peptideisosteres, and may contain amino acids other than the 20 gene-encodedamino acids. Antibodies may be modified by natural processes, such asposttranslational processing, or by chemical modification techniqueswhich are well known in the art. Such modifications are well describedin basic texts and in more detailed monographs, as well as in avoluminous research literature. Modifications can occur anywhere in theantibody, including the peptide backbone, the amino acid side-chains andthe amino or carboxyl termini, or on moieties such as carbohydrates. Itwill be appreciated that the same type of modification may be present inthe same or varying degrees at several sites in a given antibody. Also,a given antibody may contain many types of modifications. Antibodies maybe branched, for example, as a result of ubiquitination, and they may becyclic, with or without branching. Cyclic, branched, and branched cyclicantibodies may result from posttranslational natural processes or may bemade by synthetic methods. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphatidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination;see, e.g., Proteins—Structure And Molecular Properties, T. E. Creighton,W. H. Freeman and Company, New York 2nd Ed., (1993); PosttranslationalCovalent Modification Of Proteins, B. C. Johnson, Ed., Academic Press,New York, (1983) 1-12; Seifter et al., Meth. Enzymol. 182 (1990),626-646; Rattan et al., Ann. NY Acad. Sci. 663 (1992), 48-62).

The present invention also provides for fusion proteins comprising anantibody, or antigen-binding fragment, variant, or derivative thereof,and a heterologous polypeptide. In one embodiment, a fusion protein ofthe invention comprises, consists essentially of, or consists of, apolypeptide having the amino acid sequence of any one or more of the VHregions of an antibody of the invention or the amino acid sequence ofany one or more of the VL regions of an antibody of the invention orfragments or variants thereof, and a heterologous polypeptide sequence.In another embodiment, a fusion protein for use in the diagnostic andtreatment methods disclosed herein comprises, consists essentially of,or consists of a polypeptide having the amino acid sequence of any one,two, three of the V_(H)-CDRs of an antibody, or fragments, variants, orderivatives thereof, or the amino acid sequence of any one, two, threeof the V_(L)-CDRs of an antibody, or fragments, variants, or derivativesthereof, and a heterologous polypeptide sequence. In one embodiment, thefusion protein comprises a polypeptide having the amino acid sequence ofa V_(H)-CDR3 of an antibody of the present invention, or fragment,derivative, or variant thereof, and a heterologous polypeptide sequence,which fusion protein specifically binds to TTR. In another embodiment, afusion protein comprises a polypeptide having the amino acid sequence ofat least one V_(H) region of an antibody of the invention and the aminoacid sequence of at least one V_(L) region of an antibody of theinvention or fragments, derivatives or variants thereof, and aheterologous polypeptide sequence. Preferably, the V_(H) and V_(L)regions of the fusion protein correspond to a single source antibody (orscFv or Fab fragment) which specifically binds TTR. In yet anotherembodiment, a fusion protein for use in the diagnostic and treatmentmethods disclosed herein comprises a polypeptide having the amino acidsequence of any one, two, three, or more of the V_(H) CDRs of anantibody and the amino acid sequence of any one, two, three, or more ofthe V_(L) CDRs of an antibody, or fragments or variants thereof, and aheterologous polypeptide sequence. Preferably, two, three, four, five,six, or more of the V_(H)-CDR(s) or V_(L)-CDR(s) correspond to singlesource antibody (or scFv or Fab fragment) of the invention. Nucleic acidmolecules encoding these fusion proteins are also encompassed by theinvention.

Exemplary fusion proteins reported in the literature include fusions ofthe T cell receptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84(1987), 2936-2940; CD4 (Capon et al., Nature 337 (1989), 525-531;Traunecker et al., Nature 339 (1989), 68-70; Zettmeissl et al., DNA CellBiol. USA 9 (1990), 347-353; and Byrn et al., Nature 344 (1990),667-670); L-selectin (homing receptor) (Watson et al., J. Cell. Biol.110 (1990), 2221-2229; and Watson et al., Nature 349 (1991), 164-167);CD44 (Aruffo et al., Cell 61 (1990), 1303-1313); CD28 and B7 (Linsley etal., J. Exp. Med. 173 (1991),721-730); CTLA-4 (Lisley et al., J. Exp.Med. 174 (1991), 561-569); CD22 (Stamenkovic et al., Cell 66 (1991),1133-1144); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA88 (1991), 10535-10539; Lesslauer et al., Eur. J. Immunol 27 (1991),2883-2886; and Peppel et al., J. Exp. Med. 174 (1991), 1483-1489 (1991);and IgE receptor a (Ridgway and Gorman, J. Cell. Biol. 115 (1991),Abstract No. 1448).

As discussed elsewhere herein, antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention may be fused toheterologous polypeptides to increase the in vivo half-life of thepolypeptides or for use in immunoassays using methods known in the art.For example, in one embodiment, PEG can be conjugated to the antibodiesof the invention to increase their half-life in vivo; see, e.g., Leonget al., Cytokine 16 (2001), 106-119; Adv. in Drug Deliv. Rev. 54 (2002),531; or Weir et al., Biochem. Soc. Transactions 30 (2002), 512.

Moreover, antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can be fused to marker sequences,such as a peptide to facilitate their purification or detection. Inpreferred embodiments, the marker amino acid sequence is ahexa-histidine peptide (HIS), such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86 (1989), 821-824, for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37 (1984),767), GST, c-mycand the “flag” tag; see, e.g., Bill Brizzard,BioTechniques 44 (2008) 693-695 for a review of epitope taggingtechniques, and Table 1 on page 694 therein listing the most commonepitope tags usable in the present invention, the subject matter ofwhich is hereby expressly incorporated by reference.

Fusion proteins can be prepared using methods that are well known in theart; see for example U.S. Pat. Nos. 5,116,964 and 5,225,538. The precisesite at which the fusion is made may be selected empirically to optimizethe secretion or binding characteristics of the fusion protein. DNAencoding the fusion protein is then transfected into a host cell forexpression, which is performed as described hereinbefore.

Antibodies of the present invention may be used in non-conjugated formor may be conjugated to at least one of a variety of molecules, e.g., toimprove the therapeutic properties of the molecule, to facilitate targetdetection, or for imaging or therapy ofthe patient. Antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention can be labeled or conjugated either before or afterpurification, when purification is performed. In particular, antibodies,or antigen-binding fragments, variants, or derivatives thereof of theinvention may be conjugated to therapeutic agents, prodrugs, peptides,proteins, enzymes, viruses, lipids, biological response modifiers,pharmaceutical agents, or PEG.

Conjugates that are immunotoxins including conventional antibodies havebeen widely described in the art. The toxins may be coupled to theantibodies by conventional coupling techniques or immunotoxinscontaining protein toxin portions can be produced as fusion proteins.The antibodies of the present invention can be used in a correspondingway to obtain such immunotoxins. Illustrative of such immunotoxins arethose described by Byers, Seminars Cell. Biol. 2 (1991), 59-70 and byFanger, Immunol. Today 12 (1991), 51-54.

Those skilled in the art will appreciate that conjugates may also beassembled using a variety of techniques depending on the selected agentto be conjugated. For example, conjugates with biotin are prepared,e.g., by reacting a TTR-binding polypeptide with an activated ester ofbiotin such as the biotin N-hydroxysuccinimide ester. Similarly,conjugates with a fluorescent marker may be prepared in the presence ofa coupling agent, e.g. those listed herein, or by reaction with anisothiocyanate, preferably fluorescein-isothiocyanate. Conjugates of theantibodies, or antigen-binding fragments, variants or derivativesthereof of the invention are prepared in an analogous manner.

The present invention further encompasses antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention conjugatedto a diagnostic or therapeutic agent. The antibodies can be useddiagnostically to, for example, demonstrate presence of a TTRamyloidosis to indicate the risk of getting a disease or disorderassociated with misfolded, misassembled or aggregated TTR, to monitorthe development or progression of such a disease, i.e. a disease showingthe occurrence of, or related to aggregated TTR misfolded, misassembled,or as part of a clinical testing procedure to, e.g., determine theefficacy of a given treatment and/or prevention regimen. In oneembodiment thus, the present invention relates to an antibody, which isdetectably labeled. Furthermore, in one embodiment, the presentinvention relates to an antibody, which is attached to a drug. Detectioncan be facilitated by coupling the antibody, or antigen-bindingfragment, variant, or derivative thereof to a detectable substance. Thedetectable substances or label may be in general an enzyme; a heavymetal, preferably gold; a dye, preferably a fluorescent or luminescentdye; or a radioactive label. Examples of detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive materials, positronemitting metals using various positron emission tomographies, andnonradioactive paramagnetic metal ions; see, e.g., U.S. Pat. No.4,741,900 for metal ions which can be conjugated to antibodies for useas diagnostics according to the present invention. Examples of suitableenzymes include horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examplesofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ¹¹¹Inor ⁹⁹Tc. Therefore, in one embodiment the present invention provides adetectably labeled antibody, wherein the detectable label is selectedfrom the group consisting of an enzyme, a radioisotope, a fluorophoreand a heavy metal.

An antibody, or antigen-binding fragment, variant, or derivative thereofalso can be detectably labeled by coupling it to a chemiluminescentcompound. The presence of the chemiluminescent-tagged antibody is thendetermined by detecting the presence of luminescence that arises duringthe course of a chemical reaction. Examples of particularly usefulchemiluminescent labeling compounds are luminol, isoluminol, theromaticacridinium ester, imidazole, acridinium salt and oxalate ester.

One of the ways in which an antibody, or antigen-binding fragment,variant, or derivative thereof can be detectably labeled is by linkingthe same to an enzyme and using the linked product in an enzymeimmunoassay (EIA) (Voller, A., “The Enzyme Linked Immunosorbent Assay(ELISA)” Microbiological Associates Quarterly Publication, Walkersville,Md., Diagnostic Horizons 2 (1978), 1-7); Voller et al., J. Clin. Pathol.31 (1978), 507-520; Butler, Meth. Enzymol. 73 (1981), 482-523; Maggio,(ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla., (1980);Ishikawa, et al., (eds.), Enzyme Immunoassay, Kgaku Shoin, Tokyo (1981).The enzyme, which is bound to the antibody, will react with anappropriate substrate, preferably a chromogenic substrate, in such amanner as to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorimetric or by visual means. Enzymeswhich can be used to detectably label the antibody include, but are notlimited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. Additionally, the detection can be accomplished bycolorimetric methods which employ a chromogenic substrate for theenzyme. Detection may also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibody, orantigen-binding fragment, variant, or derivative thereof, it is possibleto detect the antibody through the use of a radioimmunoassay (RIA) (see,for example, Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society,(March, 1986)), which is incorporated by reference herein). Theradioactive isotope can be detected by means including, but not limitedto, a gamma counter, a scintillation counter, or autoradiography.

An antibody, or antigen-binding fragment, variant, or derivative thereofcan also be detectably labeled using fluorescence emitting metals suchas ¹⁵²Eu, or others of the lanthanide series. These metals can beattached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

Techniques for conjugating various moieties to an antibody, orantigen-binding fragment, variant, or derivative thereof are well known,see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting OfDrugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy,Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstromet al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2ndEd.), Robinson et al. (eds.), Marcel Dekker, Inc., (1987) 623-53;Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: AReview”, in Monoclonal Antibodies ‘84: Biological And ClinicalApplications, Pinchera et al. (eds.), (1985) 475-506; “Analysis,Results, And Future Prospective Of The Therapeutic Use Of RadiolabeledAntibody In Cancer Therapy”, in Monoclonal Antibodies For CancerDetection And Therapy, Baldwin et al. (eds.), Academic Press (1985)303-16, and Thorpe et al., “The Preparation And Cytotoxic Properties OfAntibody-Toxin Conjugates”, Immunol. Rev. 62 (1982), 119-158.

As mentioned, in certain embodiments, a moiety that enhances thestability or efficacy of a binding molecule, e.g., a bindingpolypeptide, e.g., an antibody or immunospecific fragment thereof can beconjugated. For example, in one embodiment, PEG can be conjugated to thebinding molecules of the invention to increase their half-life in vivo.Leong et al., Cytokine 16 (2001), 106; Adv. in Drug Deliv. Rev. 54(2002), 531; or Weir et al., Biochem. Soc. Transactions 30 (2002), 512.

VI. Compositions and Methods of Use

The present invention relates to compositions comprising theaforementioned TTR-binding molecule, e.g., antibody or antigen-bindingfragment thereof of the present invention or derivative or variantthereof, or the polynucleotide, vector, cell or peptide of the inventionas defined hereinbefore. In one embodiment, the composition of thepresent invention is a pharmaceutical composition and further comprisesa pharmaceutically acceptable carrier. Furthermore, the pharmaceuticalcomposition of the present invention may comprise further agents such asinterleukins or interferons depending on the intended use of thepharmaceutical composition. For use in the treatment of a disease ordisorder showing the occurrence of, or related to mutated, misfolded,misassembled, or aggregated TTR, such as TTR amyloidosis, the additionalagent may be selected from the group consisting of small organicmolecules, anti-TTR antibodies, and combinations thereof. Hence, in aparticular preferred embodiment the present invention relates to the useof the TTR-binding molecule, e.g., antibody or antigen-binding fragmentthereof of the present invention or of a binding molecule havingsubstantially the same binding specificities of any one thereof, thepolynucleotide, the vector or the cell of the present invention for thepreparation of a pharmaceutical or diagnostic composition forprophylactic and therapeutic treatment of a disease or disorderassociated with TTR amyloidosis, monitoring the progression of a diseaseor disorder associated with TTR amyloidosis or a response to a TTRamyloidosis treatment in a subject or for determining a subject's riskfor developing a disease or disorder associated with TTR amyloidosis.

Hence, in one embodiment the present invention relates to a methodoftreating a disease or disorder characterized by abnormal accumulationand/or deposition of TTR and/or misfolded, misassembled, aggregated,mutated TTR in affected systems and organs such as peripheral nervoussystem, autonomic nervous system, central nervous system,gastrointestinal system, vascular system especially leptomeninges,lymphoid system especially the lymphoid nodes, musculoskeletal systemespecially tendons and ligaments, the heart, eyes, kidneys, lungs, skin,tongue, thyroid gland and bladder which method comprises administeringto a subject in need thereof a therapeutically effective amount of anyone of the afore-described TTR-binding molecules, antibodies,polynucleotides, vectors, cells or peptides of the instant invention.

A particular advantage of the therapeutic approach of the presentinvention lies in the fact that the recombinant antibodies of thepresent invention are derived from B cells or memory B cells fromhealthy human subjects with no signs or symptoms of a disease, e.g.carrying an asymptomatic mutation and/or mutations, showing theoccurrence of, or related to aggregated TTR and thus are, with a certainprobability, capable of preventing a clinically manifest disease relatedto misfolded, misassembled, mutated, and/or aggregated TTR, or ofdiminishing the risk of the occurrence of the clinically manifestdisease or disorder, or of delaying the onset or progression of theclinically manifest disease or disorder. Typically, the antibodies ofthe present invention also have already successfully gone throughsomatic maturation, i.e. the optimization with respect to selectivityand effectiveness in the high affinity binding to the target TTRmolecule by means of somatic variation of the variable regions of theantibody.

The knowledge that such cells in vivo, e.g. in a human, have not beenactivated by means of related or other physiological proteins or cellstructures in the sense of an autoimmunological or allergic reaction isalso of great medical importance since this signifies a considerablyincreased chance of successfully living through the clinical testphases. So to speak, efficiency, acceptability and tolerability havealready been demonstrated before the preclinical and clinicaldevelopment of the prophylactic or therapeutic antibody in at least onehuman subject. It can thus be expected that the human anti-TTRantibodies of the present invention, both its target structure-specificefficiency as therapeutic agent and its decreased probability of sideeffects significantly increase its clinical probability of success.

The present invention also provides a pharmaceutical and diagnostic,respectively, pack or kit comprising one or more containers filled withone or more of the above described ingredients, e.g. anti-TTR antibody,binding fragment, derivative or variant thereof, polynucleotide, vector,cell and/or peptide of the present invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition oralternatively the kit comprises reagents and/or instructions for use inappropriate diagnostic assays. The composition, e.g. kit of the presentinvention is of course particularly suitable for the risk assessment,diagnosis, prevention and treatment of a disease or disorder which isaccompanied with the presence of mutated, misfolded, misassembled,and/or aggregated TTR, and in particular applicable for the treatment ofdisorders generally characterized by TTR amyloidosis comprising diseasesand/or disorders such as Familial Amyloid Polyneuropathy (FAP), FamilialAmyloid Cardiomyopathy (FAC), Senile Systemic Amyloidosis (SSA),systemic familial amyloidosis, leptomeningeal/Central Nervous System(CNS) amyloidosis including Alzheimer disease, TTR-related ocularamyloidosis, TTR-related renal amyloidosis, TTR-relatedhyperthyroxinemia, TTR-related ligament amyloidosis including carpaltunnel syndrome, rotator cuff tears and lumbar spinal stenosis, andpreeclampsia.

The pharmaceutical compositions of the present invention can beformulated according to methods well known in the art; see for exampleRemington: The Science and Practice of Pharmacy (2000) by the Universityof Sciences in Philadelphia, ISBN 0-683-306472. Examples of suitablepharmaceutical carriers are well known in the art and include phosphatebuffered saline solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions etc.Compositions comprising such carriers can be formulated by well-knownconventional methods. These pharmaceutical compositions can beadministered to the subject at a suitable dose. Administration of thesuitable compositions may be effected by different ways, e.g., byintravenous, intraperitoneal, subcutaneous, intramuscular, intranasal,topical or intradermal administration or spinal or brain delivery.Aerosol formulations such as nasal spray formulations include purifiedaqueous or other solutions of the active agent with preservative agentsand isotonic agents. Such formulations are preferably adjusted to a pHand isotonic state compatible with the nasal mucous membranes.Formulations for rectal or vaginal administration may be presented as asuppository with a suitable carrier.

The dosage regimen will be determined by the attending physician andclinical factors. As is well known in the medical arts, dosages for anyone patient depends upon many factors, including the patient's size,body surface area, age, the particular compound to be administered, sex,time and route of administration, general health, and other drugs beingadministered concurrently. A typical dose can be, for example, in therange of 0.001 to 1000 μg (or of nucleic acid for expression or forinhibition of expression in this range); however, doses below or abovethis exemplary range are envisioned, especially considering theaforementioned factors. Generally, the dosage can range, e.g., fromabout 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg (e.g., 0.02mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), ofthe host body weight. For example dosages can be 1 mg/kg body weight or10 mg/kg body weight or within the range of 1-10 mg/kg, preferably atleast 1 mg/kg. Doses intermediate in the above ranges are also intendedto be within the scope of the invention. Subjects can be administeredsuch doses daily, on alternative days, weekly or according to any otherschedule determined by empirical analysis. An exemplary treatmententails administration in multiple dosages over a prolonged period, forexample, of at least six months. Additional exemplary treatment regimensentail administration once per every two weeks or once a month or onceevery 3 to 6 months. Exemplary dosage schedules include 1-10 mg/kg or 15mg/kg on consecutive days, 30 mg/kg on alternate days or 60 mg/kgweekly. In some methods, two or more monoclonal antibodies withdifferent binding specificities are administered simultaneously, inwhich case the dosage of each antibody administered falls within theranges indicated. Progress can be monitored by periodic assessment.Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline, and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases, and the like. Furthermore, the pharmaceutical composition of theinvention may comprise further agents such as dopamine orpsychopharmacologic drugs, depending on the intended use of thepharmaceutical composition.

Furthermore, in a preferred embodiment of the present invention thepharmaceutical composition may be formulated as a vaccine, for example,if the pharmaceutical composition of the invention comprises a TTRantibody or binding fragment, derivative or variant thereof for passiveimmunization. As mentioned in the background section misfolded,misassembled, mutated and/or aggregated TTR species and/or fragments orderivatives thereof are a major trigger for TTR amyloidosis.Accordingly, it is prudent to expect that passive immunization withhuman anti-TTR antibodies and equivalent TTR-binding molecules of thepresent invention will help to circumvent several adverse effects ofactive immunization therapy concepts and lead to a reduced aggregationof TTR. Therefore, the present anti-TTR antibodies and their equivalentsof the present invention will be particularly useful as a vaccine forthe prevention or amelioration of diseases or disorders showing thepresence of, or caused by aggregated TTR such as Familial AmyloidPolyneuropathy (FAP), Familial Amyloid Cardiomyopathy (FAC), SenileSystemic Amyloidosis (SSA), systemic familial amyloidosis,leptomeningeal/Central Nervous System (CNS) amyloidosis includingAlzheimer disease, TTR-related ocular amyloidosis, TTR-related renalamyloidosis, TTR-related hyperthyroxinemia, TTR-related ligamentamyloidosis including carpal tunnel syndrome, rotator cuff tears andlumbar spinal stenosis, and preeclampsia for example.

In one embodiment, it may be beneficial to use recombinant Fab (rFab)and single chain fragments (scFvs) of the antibody of the presentinvention, which might more readily penetrate a cell membrane. Forexample, Robert et al., Protein Eng. Des. Sel. (2008); S1741-0134,published online ahead, describe the use of chimeric recombinant Fab(rFab) and single chain fragments (scFvs) of monoclonal antibody WO-2which recognizes an epitope in the N-terminal region of Abeta. Theengineered fragments were able to (i) prevent amyloid fibrillization,(ii) disaggregate preformed Abetal-42 fibrils and (iii) inhibit Abetal-42 oligomer-mediated neurotoxicity in vitro as efficiently as the wholeIgG molecule. The perceived advantages of using small Fab and scFvengineered antibody formats which lack the effector function includemore efficient passage across the blood-brain bather and minimizing therisk of triggering inflammatory side reactions. Furthermore, besidesscFv and single-domain antibodies retain the binding specificity offull-length antibodies, they can be expressed as single genes andintracellularly in mammalian cells as intrabodies, with the potentialfor alteration of the folding, interactions, modifications, orsubcellular localization of their targets; see for review, e.g., Millerand Messer, Molecular Therapy 12 (2005), 394-401.

In a different approach Muller et al., Expert Opin. Biol. Ther. (2005),237-241, describe a technology platform, so-called ‘SuperAntibodyTechnology’, which is said to enable antibodies to be shuttled intoliving cells without harming them. Such cell-penetrating antibodies opennew diagnostic and therapeutic windows. The term ‘TransMabs’ has beencoined for these antibodies.

In a further embodiment, co-administration or sequential administrationof other antibodies useful for treating a disease or disorder related tothe occurrence of mutated, misfolded, misassembled, and/or aggregatedTTR may be desirable. In one embodiment, the additional antibody iscomprised in the pharmaceutical composition of the present invention.Examples of antibodies which can be used to treat a subject include, butare not limited to, antibodies targeting CD33, SGLT2, IL-6, and IL-1.

In a further embodiment, co-administration or sequential administrationof other agents useful for treating a disease or disorder related tomisfolded, misassembled, mutated, and/or aggregated TTR, may bedesirable. In one embodiment, the additional agent is comprised in thepharmaceutical composition of the present invention. Examples of agentswhich can be used to treat a subject include, but are not limited to:Agents which stabilize the TTR-tetramer, such as Tafamidis Meglumin,diflusinal, doxycyclin with ursodeoxycholic acid; anti-inflammatoryagents such as diflusinal, corticosteroids, 2-(2,6-dichloranilino)phenylacetic acid (diclofenac), iso-butyl-propanoic-phenolic acid(ibuprofen); diuretics, Epigallocatechin gallate, Melphalanhydrochloride, dexamethasone, Bortezomib, Bortezomib-Melphalan,Bortezomib-dexamethasone, Melphalan-dexamethasone,Bortezomib-Melphalan-dexamethasone; antidepressants, antipsychoticdrugs, neuroleptics, antidementiva (e.g. the NMDA-rezeptor antagonistmemantine), acetylcholinesterase inhibitors (e.g. Donepezil, HCI,Rivastigmine, Galantamine), glutamat-antagonists and other nootropicsblood pressure medication (e.g. Dihydralazin, Methyldopa), cytostatics,glucocorticoides, angiotensin-converting-enzyme (ACE) inhibitors;anti-inflammatory agents or any combination thereof. Examples of agentswhich may be used for treating or preventing organ rejection followingclinical organ transplantation include but are not limited to the agentsof the group which lead to a weakening of the immune system, i.e.immunosuppressive comprising such as calcineurin inhibitors such ascyclosporine and Tacrolimus, inhibitors of proliferation such as mTORinhibitors comprising Everolimus and Sirolimus (rapamycin) as well asantimetabolites such as Azathioprin, Mycophenolat Mofetil/MMF andmycophenolic acid, and corticosteroids such as cortisone and cortisol aswell as synthetical substances such as Prednison or Prednisolon can beused. Additionally antibodies can be used such as anti-IL2-receptormonoclonal antibodies (e.g. Basiliximab, Daclizumab) as well as anti-CD3monoclonal antibodies (e.g. Muromonab-CD3), and polyclonal compositionssuch as anti-thymocyte globulin (ATG); and glucagon-like peptide-1(GLP-1) receptor agonists (see, e.g., Noguchi et al., Acta Med. Okayama,60 (2006), and the international application WO 2012/088157).Furthermore, additional agents might comprise agents for the prophylaxisand or treatment of infections and other side effects after an organtransplantation comprising valganciclovir, cytomegalie-immunoglobulin,gancyclovir, amphotericin B, pyrimethamin, ranitidine, ramipril,furosemide, benzbromaron. Therefore, in one embodiment a composition isprovided further comprising an additional agent useful for treating TTRamyloidosis and/or in treating or preventing organ rejection following,e.g. clinical liver transplantation. Examples of other agents that maybe used concomitant with a pharmaceutical composition of the presentinvention are described in the art; see, e.g. international applicationsWO 2009005672, WO 2010128092, WO 2012088157 or European application EP11 158 212.8.

A therapeutically effective dose or amount refers to that amount of theactive ingredient sufficient to ameliorate the symptoms or condition.Therapeutic efficacy and toxicity of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between therapeutic and toxic effects is the therapeuticindex, and it can be expressed as the ratio, LD₅₀/ED₅₀.

From the foregoing, it is evident that the present invention encompassesany use of an TTR-binding molecule and/or fragments thereof comprisingat least one CDR of the above described antibody, in particular fordiagnosing and/or treatment of a disease or disorder related to mutated,misfolded, misassembled, or aggregated TTR species and/or fragmentsthereof as mentioned above, such as TTR amyloidosis. Preferably, saidbinding molecule is an antibody of the present invention or animmunoglobulin chain thereof In addition, the present invention relatesto anti-idiotypic antibodies of any one of the mentioned antibodiesdescribed hereinbefore. These are antibodies or other binding moleculeswhich bind to the unique antigenic peptide sequence located on anantibody's variable region near the antigen-binding site and are useful,e.g., for the detection of anti-TTR antibodies in a sample obtained froma subject. In one embodiment thus, the present invention provides anantibody as defined hereinabove and below or a TTR-binding moleculehaving substantially the same binding specificities of any one thereof,the polynucleotide, the vector or the cell as defined herein or apharmaceutical or diagnostic composition comprising any one thereof foruse in prophylactic treatment, therapeutic treatment and/or monitoringthe progression or a response to treatment of a disease or disorderrelated to TTR, preferably wherein the disorder is selected from thegroup comprising Familial Amyloid Polyneuropathy (FAP), Familial AmyloidCardiomyopathy (FAC), Senile Systemic Amyloidosis (SSA), systemicfamilial amyloidosis, leptomeningeal/Central Nervous System (CNS)amyloidosis including Alzheimer disease, TTR-related ocular amyloidosis,TTR-related renal amyloidosis, TTR-related hyperthyroxinemia,TTR-related ligament amyloidosis including carpal tunnel syndrome,rotator cuff tears and lumbar spinal stenosis, and preeclampsia. Theabove group of diseases or disorders will be referred to as the group ofdisorders associated with TTR amyloidosis.

In another embodiment the present invention relates to a diagnosticcomposition comprising any one of the above described TTR-bindingmolecules, antibodies, antigen-binding fragments, polynucleotides,vectors, cells and/or peptides of the invention and optionally suitablemeans for detection such as reagents conventionally used in immuno- ornucleic acid-based diagnostic methods. The antibodies of the inventionare, for example, suited for use in immunoassays in which they can beutilized in liquid phase or bound to a solid phase carrier. Examples ofimmunoassays which can utilize the antibody of the invention arecompetitive and non-competitive immunoassays in either a direct orindirect format. Examples of such immunoassays are the radioimmunoassay(RIA), the sandwich (immunometric assay), flow cytometry, and theWestern blot assay. The antigens and antibodies of the invention can bebound to many different carriers and used to isolate cells specificallybound thereto. Examples of well-known carriers include glass,polystyrene, polyvinyl chloride, polypropylene, polyethylene,polycarbonate, dextran, nylon, amyloses, natural and modifiedcelluloses, polyacrylamides, agaroses, and magnetite. The nature of thecarrier can be either soluble or insoluble for the purposes of theinvention. There are many different labels and methods of labeling knownto those of ordinary skill in the art. Examples of the types of labelswhich can be used in the present invention include enzymes,radioisotopes, colloidal metals, fluorescent compounds, chemiluminescentcompounds, and bioluminescent compounds; see also the embodimentsdiscussed hereinabove.

By a further embodiment, the TTR-binding molecules, in particularantibodies of the present invention may also be used in a method for thediagnosis of a disease or disorder in an individual by obtaining a bodyfluid sample from the tested individual which may be a blood sample, aplasma sample, a serum sample, a lymph sample or any other body fluidsample, such as a saliva or a urine sample and contacting the body fluidsample with an antibody of the instant invention under conditionsenabling the formation of antibody-antigen complexes. The level of suchcomplexes is then determined by methods known in the art, a levelsignificantly higher than that formed in a control sample indicating thedisease or disorder in the tested individual. In the same manner, thespecific antigen bound by the antibodies of the invention may also beused. Thus, the present invention relates to an in vitro immunoassaycomprising the binding molecule, e.g., antibody or antigen-bindingfragment thereof of the invention.

In a further embodiment of the present invention the TTR-bindingmolecules, in particular antibodies of the present invention may also beused in a method for the diagnosis of a disease or disorder in anindividual by obtaining a biopsy from the tested individual which may beskin, salivary gland, hair roots, heart, colon, nerve, subcutaneous fatbiopsies, or a biopsy from any affected organs.

In this context, the present invention also relates to meansspecifically designed for this purpose.

For example, an antibody-based array may be used, which is for exampleloaded with antibodies or equivalent antigen-binding molecules of thepresent invention which specifically recognize TTR. Design of microarrayimmunoassays is summarized in Kusnezow et al., Mol. Cell Proteomics 5(2006), 1681-1696. Accordingly, the present invention also relates tomicroarrays loaded with TTR-binding molecules identified in accordancewith the present invention.

In one embodiment, the present invention relates to a method ofdiagnosing a disease or disorder related to mutated, misfolded,misassembled and/or aggregated TTR species and/or fragments thereof in asubject, the method comprising determining the presence of TTR and/ormisfolded, misassembled or aggregated TTR in a sample from the subjectto be diagnosed with at least one antibody of the present invention, aTTR-binding fragment thereof or an TTR-binding molecule havingsubstantially the same binding specificities of any one thereof, whereinthe presence of pathologically mutated, misfolded, misassembled oraggregated TTR is indicative for TTR amyloidosis and an increase of thelevel of the pathologically misfolded, misassembled or aggregated TTR incomparison to the level of the physiological TTR is indicative forprogression of TTR amyloidosis in said subject.

The subject to be diagnosed may be asymptomatic or preclinical for thedisease. Preferably, the control subject has a disease associated withmisfolded, misassembled or aggregated TTR, e.g. Familial AmyloidPolyneuropathy (FAP), Familial Amyloid Cardiomyopathy (FAC) or SenileSystemic Amyloidosis (SSA), wherein a similarity between the level ofpathologically misfolded, misassembled or aggregated TTR and thereference standard indicates that the subject to be diagnosed has a TTRamyloidosis or is at risk to develop a TTR amyloidosis. Alternatively,or in addition as a second control the control subject does not have aTTR amyloidosis, wherein a difference between the level of physiologicalTTR and/or of misfolded, misassembled or aggregated TTR and thereference standard indicates that the subject to be diagnosed has a TTRamyloidosis or is at risk to develop a TTR amyloidosis. Preferably, thesubject to be diagnosed and the control subject(s) are age-matched. Thesample to be analyzed may be any body fluid suspected to containpathologically misfolded, misassembled or aggregated TTR, for example ablood, blood plasma, blood serum, urine, peritoneal fluid, saliva orcerebral spinal fluid (CSF).

The level of physiological TTR and/or of pathologically misfolded,misassembled or aggregated TTR may be assessed by any suitable methodknown in the art comprising, e.g., analyzing TTR by one or moretechniques chosen from Western blot, immunoprecipitation, enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescentactivated cell sorting (FACS), two-dimensional gel electrophoresis, massspectroscopy (MS), matrix-assisted laser desorption/ionization-time offlight-MS (MALDI-TOF), surface-enhanced laser desorption ionization-timeof flight (SELDI-TOF), high performance liquid chromatography (HPLC),fast protein liquid chromatography (FPLC), multidimensional liquidchromatography (LC) followed by tandem mass spectrometry (MS/MS), andlaser densitometry. Preferably, said in vivo imaging of TTR comprisesscintigraphy, positron emission tomography (PET), single photon emissiontomography (SPECT), near infrared (NIR) optical imaging or magneticresonance imaging (MRI). In a further aspect, the present inventionrelates to the diagnosis of TTR amyloidosis, monitoring the treatment ofthis disease and determining the diagnostic or therapeutic utility of ananti-TTR drug in a tissue- and biopsy-free, i.e. non-invasive method.

Normally, the concentration of TTR aggregates and/or misfolded TTR whichcan be detected in a body fluid, for example blood plasma is very lowand thus the diagnosis of TTR amyloidosis is burdensome andtime-consuming. In particular, the diagnosis of TTR amyloidosis diseasesis a difficult and lengthy process, since various diseases present verysimilar signs and symptoms, such that the formal diagnosis of FamilialAmyloid Polyneuropathy (FAP), Familial Amyloid Cardiomyopathy (FAC) andSenile Systemic Amyloidosis (SSA) typically requires collection oftissue biopsies and identification of TTR amyloid deposits by means ofcomplex histological staining techniques. As tissue biopsies are verysmall and TTR amyloid deposits dispersed, histological determination ofTTR amyloidosis is typically associated with high frequency of falsenegative results and delays for the patients.

However, in accordance with the present invention it could surprisinglybe shown that after a single administration of a subject anti-TTRantibody a measurement of aggregates and/or misfolded TTR bound toanti-TTR antibodies in blood was possible; see Example 13 and FIG. 14.Therefore, thanks to the probably unique property of the anti-TTRantibody of the present invention to remove TTR from amyloidogenic TTRdeposits and transport into blood a novel method of diagnosing disordersassociated with misfolded, mutated, and/or aggregated TTR in a patientor subject has been developed, which method has the potential to replacetissue biopsy and histological analysis in the diagnostic process of TTRamyloid diseases. The method relies on the use of an antibody specificfor the pathological conformation of TTR protein, which is injected tothe patient and used to probe for the presence of misfolded and/oraggregated TTR protein anywhere in patient's body. After a short delay,for example 2 days, following antibody injection in a patient, a bloodsample is drawn and used to detect if the antibody has captured anddetached misfolded and/or aggregated TTR particles from TTR deposits.The major advantage of this method compared to histology has to do withthe injection of the anti-TTR antibody directly in patients, where bloodcirculation allows its circulation through every tissue and organ, anddetection of misfolded and/or aggregated TTR protein depositsindependently of their localization.

Thus, in a further aspect the present invention relates to a method ofdiagnosing a disease associated with TTR amyloidosis comprising assayingthe level of misfolded and/or aggregated TTR in a sample from a subjectfollowing administration of an anti-TTR antibody to the subject, whereinthe presence or elevated the level of misfolded and/or aggregated TTR inthe sample of the subject compared to the control such as a sampleobtained from the subject prior to administration of the anti-TTRantibody indicates a disease associated with TTR amyloidosis.Furthermore, since as shown in Example 13 the novel method is alsouseful for characterizing anti-TTR drugs and the course of treatment ofTTR amyloidosis, respectively, the novel method of the present inventionis also intended for monitoring the treatment of the disease with ananti-TTR antibody or determining the diagnostic or therapeutic utilityof an anti-TTR antibody. In this context, the person skilled in the artwill recognize that the method of the present invention is not limitedto the investigation of the therapeutic utility and efficacy of anti-TTRantibodies but also applicable to other kinds of anti-TTR drugs whichare capable of degrading TTR amyloid deposits. For example, an anti-TTRantibody of the present invention may be administered in conjunctionwith another anti-TTR drug and the level of misfolded and/or aggregatedTTR in the sample of the subject having been treated is compared to acontrol obtained from the subject prior to administration of both theanti-TTR antibody and the anti-TTR drug but only after anti-TTR antibodytreatment.

In one preferred embodiment of the present invention, in particular whenusing non-human animals for testing recombinant human-derived antibodiesas illustrated in Example 13 and other anti-TTR antibodies intended foruse in humans in general the level of misfolded and/or aggregated TTR inthe sample is assayed by determining a complex formed between theanti-TTR antibody and the misfolded and/or aggregated TTR, for exampleby immuno-precipitation with an anti-human IgG or anti-idiotypicantibody. Alternatively, a second anti-TTR antibody may be used whichrecognizes TTR at an epitope different substantially different from theepitope of the drug candidate anti-TTR antibody so as to bind thecomplex formed by the drug candidate anti-TTR antibody and TTR and thusdetected its presence, for example by way of ELISA orimmune-precipitation.

With respect to the diagnostic aspect in particular for a human subjectand patient, the presence and elevated level of misfolded and/oraggregated TTR and complex thereof with the anti-TTR antibody,respectively, indicates the presence of TTR amyloid deposits in thehuman body, for example in the heart, peripheral nervous system (PNS),eyes, muscles, gastro-intestinal tract, kidneys, vascular system and thecentral nervous system (CNS) of a patient or subject. Thus, the methodof the present invention allows the identification and determination ofa disease associated with TTR amyloidosis in the subject's body on theone hand and removal of TTR deposits from patient's body on the other,thereby also indicating the therapeutic progress of a given treatmentand efficacy of a TTR amyloidosis specific drug such as an anti-TTRantibody.

Hence, as demonstrated in Example 13 the anti-TTR antibody of thepresent invention is capable of binding misfolded and/or aggregated TTRwith sufficient affinity to alter the stability of pathological TTRdeposits such as to capture and remove misfolded and/or aggregated TTRfrom the deposits into a body fluid, in particular blood.

The anti-TTR antibody to be used in accordance with the method of thepresent invention may be any TTR antibody which is specific for thepathological conformation of TTR, i.e. misfolded, mutated, and/oraggregated TTR. However, in a preferred embodiment the anti-TTR antibodyutilized in the tissue-free method is an anti-TTR antibody orTTR-binding molecule of the present invention described herein andillustrated in the Examples.

In this context, the anti-TTR antibody may be modified and for exampleattached to a detectable label as described for any of the otherembodiments hereinbefore. In addition, immunoassays such as westernblot, dot blot, (sandwich) ELISA and the like known in the art anddescribed for other diagnostic methods and uses based on the anti-TTRantibody and peptide of the present invention may be adapted to thenovel TTR amyloid assay of the present invention.

As shown in Example 13 and FIG. 14 using the TTR amyloid assay it couldbe shown that anti-TTR antibodies of the present invention are capableof capturing and detaching misfolded and/or aggregated TTR from TTRamyloid deposits and the corresponding immuno-complex can be measured ina sample of body fluid, in particular blood of the patient or subject;see Example 13 and FIG. 14. Accordingly, in one embodiment of thepresent invention the anti-TTR antibody can bind misfolded and/oraggregated TTR with sufficient affinity to alter the net efflux of themisfolded and/or aggregated TTR from e.g. heart, peripheral nervoussystem (PNS), eyes, muscles, gastro-intestinal tract, kidneys, vascularsystem and the central nervous system (CNS).

The body fluid sample, preferably blood or CSF from the subject, whereincaptured and detached misfolded and/or aggregated TTR bound to theanti-TTR antibody is present, is obtained at a specified time intervalfollowing administration. This specified time interval followingadministration is typically less than one week. In a preferredembodiment this time interval after administration of the anti-TTRantibody is less than or equal to 48 hours.

As mentioned supra, the tissue-free method described supra, can also beutilized to determine the success of the treatment, i.e. by measurementof misfolded and/or aggregated TTR species captures by anti-TTRantibodies before and after treatment. Thus, in a further or additionalembodiment, the tissue-free method of the present invention may furthercomprise the comparison between the level of the misfolded and/oraggregated TTR in the sample of a body fluid to a sample obtained fromthe subject prior to administration of an anti-TTR antibody.Accordingly, in one embodiment the method of the present invention isused to determine the effectiveness of a treatment of TTR amyloidosis orfor monitoring the progression of a disease or condition associated withpathological TTR in a patient or subject.

As mentioned, samples of subjects utilized in the methods describedabove can be obtained before or after administration of an anti-TTRantibody. However, samples can also be obtained from medical facilitiesor practicing physicians as well as other institutions from whichclinical samples from a subject can be obtained. The facilities,physicians, etc. can not only perform the administration of an anti-TTRantibody to the subject and the collection of appropriate samples foruse in the above method, but monitor and/or the treatment of thepatient, i.e. by varying the amount, time, frequency of administrationof the antibody, interrupting a therapy, replace or combine the anti-TTRantibody by at least another anti-TTR antibody or therapeutic agent. Thelevel of TTR can be assessed by any suitable method known in the art.Methods suitable are described below and in international application WO2013/066818, the disclosure content of which is incorporated herein byreference.

In one aspect of the present invention, an antibody of the presentinvention or a TTR-binding molecule having substantially the samebinding specificities of any one thereof, the polynucleotide, the vectoror the cell as defined hereinabove or a pharmaceutical or diagnosticcomposition comprising any one thereof is provided for use inprophylactic treatment, therapeutic treatment, and/or monitoring theprogression or a response to treatment of a disease or disorder relatedto TTR. In general thus, the present invention also relates to a methodof diagnosing or monitoring the progression of a disease or disorderrelated to TTR (such as TTR amyloidosis) in a subject, the methodcomprising determining the presence of TTR in a sample from the subjectto be diagnosed with at least one antibody of the present invention or aTTR-binding molecule having substantially the same binding specificitiesof any one thereof, wherein the presence of mutated, misfolded,misassembled or aggregated TTR species or fragments thereof isindicative for the disease or disorder. In one embodiment said method ofdiagnosing or monitoring the progression of TTR amyloidosis in a subjectis provided, the method comprising determining the presence of mutated,misfolded, misassembled or aggregated TTR and/or fragments thereof in asample from the subject to be diagnosed with at least one antibody ofthe present invention or a TTR-binding molecule having substantially thesame binding specificities of any one thereof, wherein the presence ofmutated, misfolded, misassembled or aggregated TTR and/or fragmentthereof is indicative of presymptomatic, prodromal or clinical TTRamyloidosis an increase of the level of TTR oligomers, aggregates orfibrils in comparison to the level of the physiological TTR or incomparison to a reference sample derived from a healthy control subjector a control sample from the same subject is indicative for progressionof presymptomatic, prodromal or established TTR amyloidosis. It would beappreciated by any person skilled in the art that in one embodiment saidmethod is used as well for the diagnosing or monitoring the progressionof any other disease or disorder from the group of disorders related toTTR as defined hereinabove.

As indicated above, the antibodies of the present invention, fragmentsthereof and molecules of the same binding specificity as the antibodiesand fragments thereof may be used not only in vitro but in vivo as well,wherein besides diagnostic, therapeutic applications as well may bepursued. In one embodiment thus, the present invention also relates to aTTR binding molecule comprising at least one CDR of an antibody of thepresent invention for the preparation of a composition for in vivodetection/imaging of or targeting a therapeutic and/or diagnostic agentto TTR in the human or animal body. Potential therapeutic and/ordiagnostic agents may be chosen from the nonexhaustive enumerations ofthe therapeutic agents useful in treatment TTR amyloidosis and potentiallabels as indicated hereinbefore. In respect of the in vivo imaging, inone preferred embodiment the present invention provides said TTR bindingmolecule comprising at least one CDR of an antibody of the presentinvention, wherein said in vivo imaging comprises scintigraphy, positronemission tomography (PET), single photon emission tomography (SPECT),near infrared (NIR) optical imaging or magnetic resonance imaging (MRI).In a further embodiment the present invention also provides saidTTR-binding molecule comprising at least one CDR of an antibody of thepresent invention, or said molecule for the preparation of a compositionfor the above specified in vivo imaging methods, for the use in themethod of diagnosing or monitoring the progression of a disease ordisorder related to TTR in a subject, as defined hereinabove.

VII. Peptides with Aggregation Specific TTR Epitopes

In a further aspect the present invention relates to peptides having anepitope of TTR specifically recognized by any antibody of the presentinvention. Preferably, such peptide comprises or consists of an aminoacid sequence as indicated in SEQ ID NO: 49, SEQ ID NO: 50, or in SEQ IDNO: 51 as the unique linear epitope recognized by the antibody or amodified sequence thereof in which one or more amino acids aresubstituted, deleted and/or added, wherein the peptide is recognized byany antibody of the present invention, preferably by antibodyNI-301.59F1, NI-301.35G11, NI-301.37F1, or NI-301.12D3.

In one embodiment of this invention such a peptide may be used fordiagnosing or monitoring a disease or disorder related to misfolded,misassembled or aggregated TTR species and/or fragment thereof in asubject, such as TTR amyloidosis comprising a step of determining thepresence of an antibody that binds to a peptide in a biological sampleof said subject, and being used for diagnosis of such a disease in saidsubject by measuring the levels of antibodies which recognize the abovedescribed peptide of the present invention and comparing themeasurements to the levels which are found in healthy subjects ofcomparable age and gender. Thus in one embodiment the present inventionrelates to a method for diagnosing TTR amyloidosis indicative ofpresymptomatic or clinical FAP and/or FAC in a subject, comprising astep of determining the presence of an antibody that binds to a peptideas defined above in a biological sample of said subject. According tothis method, an elevated level of measured antibodies specific for saidpeptide of the present invention is indicative for diagnosing in saidsubject presymptomatic or clinical FAP and/or FAC or for diagnosing insaid subject any other disease or disorder from the group of disordersrelated to TTR as defined hereinabove. Furthermore, since the peptide ofthe present invention contains an epitope of a therapeutically effectiveantibody derived from a human such peptide can of course be used as anantigen, i.e.an immunogen for eliciting an immune response in a subjectand stimulating the production of an antibody of the present inventionin vivo. The peptide of the present invention may be formulated in anarray, a kit and composition such as a vaccine, respectively, asdescribed hereinbefore. In this context, the present invention alsorelates to a kit useful in the diagnosis or monitoring the progressionof TTR amyloidosis, said kit comprising at least one antibody of thepresent invention or a TTR-binding molecule having substantially thesame binding specificities of any one thereof, the polynucleotide, thevector or the cell and/or the peptide as respectively definedhereinbefore, optionally with reagents and/or instructions for use.

These and other embodiments are disclosed and encompassed by thedescription and examples of the present invention. Further literatureconcerning any one of the materials, methods, uses, and compounds to beemployed in accordance with the present invention may be retrieved frompublic libraries and databases, using for example electronic devices.For example the public database “Medline” may be utilized, which ishosted by the National Center for Biotechnology Information (NCBI)and/or the National Library of Medicine at the National Institutes ofHealth (NLM.NIH). Further databases and web addresses, such as those ofthe European Bioinformatics Institute (EBI), which is part of theEuropean Molecular Biology Laboratory (EMBL) are known to the personskilled in the art and can also be obtained using internet searchengines. An overview of patent information in biotechnology and a surveyof relevant sources of patent information useful for retrospectivesearching and for current awareness is given in Berks, TIBTECH 12(1994), 352-364.

The above disclosure generally describes the present invention. Unlessotherwise stated, a term as used herein is given the definition asprovided in the Oxford Dictionary of Biochemistry and Molecular Biology,Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 019 850673 2. Several documents are cited throughout the text of thisspecification. Full bibliographic citations may be found at the end ofthe specification immediately preceding the claims. The contents of allcited references (including literature references, issued patents,published patent applications as cited throughout this applicationincluding the background section and manufacturer's specifications,instructions, etc.) are hereby expressly incorporated by reference;however, there is no admission that any document cited is indeed priorart as to the present invention. A more complete understanding can beobtained by reference to the following specific examples which areprovided herein for purposes of illustration only and are not intendedto limit the scope of the invention.

EXAMPLES Example 1 Isolation and Identification of Anti-TTR Antibodies

Human-derived antibodies targeting TTR and/or mutated, misfolded,misassembled, and/or aggregated TTR species and/or fragments thereofwere identified utilizing the method described in the internationalapplication WO 2008/081008, the disclosure content of which isincorporated herein by reference, with modifications. In particular,human wild-type TTR protein obtained by purification from human plasma,and wild-type and mutant TTR proteins obtained by recombinant expressionwere used in both native and misfolded-aggregated conformations for theidentification of TTR-targeting antibodies. The misfolded-aggregatedconformations were produced in vitro under acidic conditions, using aprocedure similar to the one described in Colon W. et al, Biochemistry,31 (1992), 8654-8660, with minor modifications.

Example 2 Determination of Antibody Sequence

The amino acid sequences of the variable regions of the above identifiedanti-TTR antibodies were determined on the basis of their mRNAsequences, see FIG. 1. In brief, living B cells of selectednon-immortalized memory B cell cultures were harvested. Subsequently,the mRNAs from cells producing selected anti-TTR antibodies wereextracted and converted in cDNA, and the sequences encoding theantibody's variable regions were amplified by PCR, cloned into plasmidvectors and sequenced. In brief, a combination of primers representingall sequence families of the human immunoglobulin germline repertoirewas used for the amplifications of leader peptides, V-segments andJ-segments. The first round of amplification was performed using leaderpeptide-specific primers in 5′-end and constant region-specific primersin 3′-end (Smith et al., Nat Protoc. 4 (2009), 372-384). For heavychains and kappa light chains, the second round of amplification wasperformed using V-segment-specific primers at the 5′-end andJ-segment-specific primers at the 3′-end. For lambda light chains, thesecond round amplification was performed using V-segment-specificprimers at the 5′-end and a C-region-specific primer at the 3′-end(Marks et al., Mol. Biol. 222 (1991), 581-597; de Haard et al., J. Biol.Chem. 26 (1999), 18218-18230).

Identification of the antibody clone with the desired specificity wasperformed by re-screening on ELISA upon recombinant expression ofcomplete antibodies. Recombinant expression of complete human IgG1antibodies was achieved upon insertion of the variable heavy and lightchain sequences “in the correct reading frame” into expression vectorsthat complement the variable region sequence with a sequence encoding aleader peptide at the 5′-end and at the 3′-end with a sequence encodingthe appropriate constant domain(s). To that end the primers containedrestriction sites designed to facilitate cloning of the variable heavyand light chain sequences into antibody expression vectors. Heavy chainimmunoglobulins were expressed by inserting the immunoglobulin heavychain RT-PCR product in frame into a heavy chain expression vectorbearing a signal peptide and the constant domains of human or mouseimmunoglobulin gamma 1. Kappa light chain immunoglobulins were expressedby inserting the kappa light chain RT-PCR-product in frame into a lightchain expression vector providing a signal peptide and the constantdomain of human kappa light chain immunoglobulin. Lambda light chainimmunoglobulins were expressed by inserting the lambda light chainRT-PCR-product in frame into a lambda light chain expression vectorproviding a signal peptide and the constant domain of human or mouselambda light chain immunoglobulin.

Functional recombinant monoclonal antibodies were obtained uponco-transfection into HEK 293 or CHO cells (or any other appropriaterecipient cell line ofhuman or mouse origin) of an Ig-heavy-chainexpression vector and a kappa or lambda Ig-light-chain expressionvector. Recombinant human monoclonal antibody was subsequently purifiedfrom the conditioned medium using a standard Protein A columnpurification. Recombinant human monoclonal antibody can produced inunlimited quantities using either transiently or stably transfectedcells. Cell lines producing recombinant human monoclonal antibody can beestablished either by using the Ig-expression vectors directly or byre-cloning of Ig-variable regions into different expression vectors.Derivatives such as F(ab), F(ab)2 and scFv can also be generated fromthese Ig-variable regions.

The framework and complementarity determining regions were determined bycomparison with reference antibody sequences available in databases suchas Abysis (http://www.bioinf.org.uk/abysis/), and annotated using theKabat numbering scheme (http://www.bioinf.org.uk/abs/). The amino acidsequences of the variable regions of the subject antibodies NI-301.59F1,NI-301.35G11, NI-301.37F1, NI-301.2F5, NI-301.28B3, NI-301.119C12,NI-301.5D8, NI-301.9D5, NI-301.104F5, NI-301.21F10, NI-301.9G12,NI-301.12D3, NI-301.37F1-PIMC, NI-301.44E4, NI-301.18C4, NI-301.11A10,NI-301.3C9, NI-301.14D8, NI-301.9X4, and NI-301.14C3 includingindication of the framework (FR) and complementarity determining regions(CDRs) are shown in FIG. 1A-1T.

In the following, the high affinity of the subject antibodies tomisfolded-aggregated TTR conformations and substantial lack of bindingto native wild-type TTR conformations, thereby demonstrating a strongselectivity for mutant, misfolded, misassembled and/or aggregated TTR isexemplary illustrated for antibodies NI-301.59.F1, NI-301.35G11, andNI-301.37F1. However, preliminary experiments for other subjectantibodies suggest substantially the same preferential binding tomutant, misfolded, misassembled and/or aggregated TTR over physiologicalTTR species like antibodies NI-301.59.F1, NI-301.35G11, and NI-301.37F1.

Example 3 Binding Affinity of Anti-TTR Antibodies Utilizing Direct ELISAand EC50 Determination

The antibody capacity to bind TTR and/or misfolded, misassembled and/oraggregated forms of TTR was evaluated by means of direct ELISA assays atvarying antibody concentrations. This allows to determinate for eachantibody its half maximal effective concentration (EC50) in this assay,which is a commonly used proxy for the antibody binding affinity, seeFIG. 2. In brief, ELISA plates (high-bind, clear polystyrene, half-area,flat bottom) were coated with misfolded-aggregated human wild-type TTR,misfolded-aggregated recombinant V30M-TTR (both prepared as described inthe Example 1) and bovine serum albumin (BSA) at a concentration of 10μg/ml in phosphate buffer saline (PBS) for 1 h at 37° C., andsubsequently blocked with a solution of 2% BSA and 0.1% tween-20 in PBS(PBS-T) for 1 h at room temperature (RT). Antibodies against TTR werediluted in PBS at 11 different concentrations ranging from 4 to 400 nM,and incubated in the ELISA plates overnight at 4° C. After 3 washes withPBS-T, ELISA plates were incubated with a HRP-coupled, humanIgG-specific secondary antibody for 1 h at RT (1/4000 dilution). After 3washes with PBS-T, the ELISA reactions were developed with TMB forexactly 10 min at RT and quantified by measuring the optical density at450 nm (OD450 nm).

The exemplary antibodies NI-301.59F1, NI-301.35G11, and NI-301.37F1exhibited strong binding to misfolded-aggregated wild-type and mutantTTR, but not to the control BSA, see FIG. 2 A-C. Subsequently, theantibody's EC50s were determined by fitting the data with a non-linearregression using the least square method in order to estimate theantibody binding affinity under these conditions.

The exemplary antibodies NI-301.59F1, NI.35G11, and NI-301.37F1exhibited high affinity for misfolded-aggregated human wild-type TTRcorresponding to EC5Os of 3.0 nM, 3.9 nM, and 0.35 nM respectively. Theexemplary antibodies also exhibited high affinity formisfolded-aggregated recombinant mutant V30M-TTR corresponding to EC50sof 15.5 nM, 5.0 nM, and 0.15 nM respectively.

Example 4 Binding Selectivity of Anti-TTR Antibodies Utilizing Dot Blot

To evaluate the binding selectivity of the TTR-antibodies and/orfragments thereof for native or misfolded, misassembled and/oraggregated TTR conformations, human wild-type TTR protein in native ormisfolded-aggregated conformations and recombinant V30M-TTR protein inmisfolded-aggregated conformations were diluted in PBS at 4 differentconcentrations, and deposited by vacuum filtration on a nitrocellulosemembrane. The membrane was briefly dried (10 min) and blocked with 3%milk in PBS-T for 1 h at RT, and subsequently incubated with anti-TTRantibodies overnight at 4° C. After 3 washes with PBS-T for 5 min at RT,the membrane was incubated with the appropriate secondary antibody(HRP-coupled; 1/10000 dilution) for 1 h at RT. After 3 washes withPBS-T, the membrane was developed with luminol and the signal intensityquantified by measuring luminescence.

The exemplary commercial anti-TTR antibody bound to native as well asmisfolded-aggregated TTR conformations with similar affinity, therebydemonstrating its absence of binding selectivity for native ormisfolded, misassembled and/or aggregated TTR conformations, see FIG. 3A. In contrast, the exemplary antibodies NI-301.59.F1, NI-301.35G11, andNI-301.37F1 bound with high affinity to misfolded-aggregated TTRconformations only, and showed no binding to native TTR conformations,thereby demonstrating a strong selectivity for misfolded, misassembledand/or aggregated TTR (FIG. 3 B2, C2, D2). Accordingly, the antibodiesNI-301.35G11 and NI-301.37F1 also showed strong binding to themisfolded-aggregated recombinant V30M-TTR protein, as shown in FIGS. 3C3 and D3.

To further characterize the antibody binding selectivity, various TTRpreparations including wild-type and mutant, native andmisfolded-aggregated conformations, and a collection of 12 human plasmasamples were processed similarly for analysis by dot blot, using murinechimeric anti-TTR antibodies and HRP-coupled, anti-mouse IgG2a secondaryantibody for detection (FIG. 6).

The commercial antibody exhibited strong binding to all TTR preparation,including wild-type and mutant, native and misfolded-aggregated TTRpreparations, and was able to detect TTR in all human plasma samples.This further demonstrates the absence of selectivity for native oraggregated conformations, see FIG. 6 A. In contrast, the exemplary mousechimeric antibody NI-301.mur35G11 exhibited very strong binding to themisfolded-aggregated wild-type TTR sample (FIG. 6 C1), and also strongbinding to the mutantV30M-TTR protein (FIG. 6 C4), and to the mutantY78F-TTR protein (FIG. 6 C6). However, the NI-301.mur35G11 antibody didnot bind to TTR in the human plasma samples. This further demonstratesthe strong selectivity of NI-301.mur35G11 for mutated, misfolded,misassembled and/or aggregated TTR protein.

Example 5 Binding Specificity and Selectivity of Anti-TTR AntibodiesUtilizing Western Blot

The binding specificity and selectivity of anti-TTR antibodies wasevaluated by means of western blot, see FIG. 4. In brief, humanwild-type TTR protein (300 ng) in native or misfolded-aggregatedconformations, and wild-type mouse liver extract (10 μg total protein)were loaded on a SDS-PAGE gel and transferred onto a nitrocellulosemembrane using a semi-dry transfer system. The membrane was subsequentlyblocked with 2% BSA in PBS-T for 1 h at RT, and incubated overnight at4° C. with anti-TTR antibodies diluted in blocking buffer. After 4washes with PBS-T for 5 min at RT, the membrane was incubated with theappropriate secondary antibody (HRP-coupled; 1/10000 dilution inblocking buffer) for 1 h at RT. After 3 washes with PBS-T and a finalone in PBS, the membrane was developed with luminol and the signalintensity quantified by measuring luminescence. Shortly before use, themisfolded-aggregated TTR sample was submitted to crosslinking withglutaraldehyde (1%, 5 min, 37° C.) to prevent the dissociation of TTRaggregates during the preparation process for SDS-PAGE. In contrast, thenative TTR sample was not crosslinked before use, such that the TTRhomotetramer (which is the native TTR conformation under physiologicalconditions) almost entirely dissociated into monomers and dimers.

The commercial anti-TTR antibody showed very strong binding to the TTRmonomers and dimers of the human native TTR sample (FIG. 4 A1), and asimilarly strong binding to cross-linked misfolded-aggregated TTR sample(FIG. 4 A2), thereby demonstrating the absence of selectivity for nativeor misfolded, misassembled and/or aggregated TTR conformations. Incontrast, the exemplary anti-TTR antibodies NI-301.59F1, NI-301.35G11,and NI-301.37F1 showed very strong binding to the cross-linkedmisfolded-aggregated TTR sample (FIG. 4 B2, C2, D2) but no binding atall to the TTR monomers and dimers of the human native TTR sample (FIG.4 B1, C1, D1), thereby demonstrating strong selectivity for misfolded,misassembled and/or aggregated TTR conformations over native TTRconformations.

In addition to that, the commercial and the exemplary anti-TTRantibodies had very low levels of binding to the proteins contained inthe mouse liver extract (FIG. 4 A3, B3, C3, D3). In view of the highamount of liver proteins used for the experiment and the high antibodyconcentrations with respect to their binding affinity, this indicatesthat the exemplary antibodies have a remarkable specificity for TTR anddo not bind significantly to other proteins. Furthermore, it appearsthat the exemplary antibodies do not bind to the mouse TTR proteincontained at high levels in the mouse liver extract, indicating that theexemplary antibodies show specificity for the human misfolded TTRprotein. However, the epitope of the antibody NI-301.37F1 is present onthe TTR protein of rat and mouse. Accordingly, the primary amino acidsequence of the epitope may not be necessarily decisive for thedetection of misfolded TTR, but the conformation.

To further characterize the antibody binding capacity, or its absencethereof, to native TTR protein, the exemplary antibodies were evaluatedfor their capacity to bind to the TTR protein contained in human plasmasamples using the same western blot technique as described here above,(FIG. 5). The only technical difference consisted in trimming the upperpart of the gel at about 25-30 kDa, and using only the lower part of thegel for transfer of the proteins onto the nitrocellulose membrane. Thisis to eliminate the heavy and light chains of the human antibodiespresent at high concentration in the plasma samples, which couldpotentially interfere with the analysis.

In contrast with the commercial antibody used as reference, theexemplary antibodies NI-301.35G11 and NI-301.37F1 did not detect at allthe human TTR protein contained in human plasma samples, therebyindicating binding selectivity for a TTR conformation which in notpresent in the analyzed samples under these conditions.

Example 6 Binding Selectivity of Anti-TTR Antibodies in SolutionUtilizing Immunoprecipitation

To further verify the binding selectivity of the anti-TTR antibodies ofthe present invention, human wild-type and recombinant TTR protein innative and misfolded-aggregated conformations, and a human plasma sampleat 3 different dilutions in PBS were used for TTR immunoprecipitation(IP). In brief, protein-A coated magnetic beads were incubated withanti-TTR antibodies diluted in manufacturer binding buffer for 30 min atRT. The antibody/protein A complex was retrieved and incubated overnightat 4° C. with TTR preparations and human plasma samples. After washes,the antibody/protein A complex was resuspended in SDS loading buffer,heated 5 min at 90° C. and processed for western blot analysis.

As shown in FIG. 7 the exemplary TTR antibodies NI-301.35G11 andNI-301.37F1 showed in contrast to the commercial TTR antibody Dako A0002no binding to the plasma samples (FIG. 7 A7-9, B7-9, C7-9), as well asno binding to the native wild-type and recombinant TTR samples (FIG. 7B3, C3, B5, C5). However, a strong binding was assessed in the sample,wherein misfolded-aggregated forms of TTR were present (FIG. 7 B4, C4,B6, C6).

These results indicate that the exemplary antibodies NI-301.35G11 andNI-301.37F1 are able to bind misfolded, misassembled and/or aggregatedTTR conformations in solution, and show remarkable selectivity for theseconformations.

Example 7 Binding to Pathological TTR Aggregates in FAP Mouse Tissue

Exemplary anti-TTR antibodies were evaluated by immunohistochemistry(IHC) for their capacity to bind pathological and non-pathological TTRprotein as present in the tissues of transgenic mice expressingexclusively the human V30M-TTR protein and not the mouse TTR protein(thereafter named FAP mice). These antibodies were also evaluated fornon-specific binding on tissues from TTR knockout (TTR-KO) mice notexpressing any TTR protein, and the corresponding transgenic andknockout mouse lines were initially generated and described by Prof.Suichiro Maeda (Kohno K. et al., American Journal of Pathology 140(4)(1997), 1497-1508). In brief, immunohistochemistry was performed onparaffin embedded mouse tissues cut in 3-5 μm thick sections. Sectionswere initially dewaxed and rehydrated, and treated with 3% H2O2 inmethanol for 20 min at RT. Blocking buffer (PBS+5% serum (horse/goat)+4%BSA) was applied for 1 h at RT, and replaced with anti-TTR antibodydiluted in PBS for overnight incubation at 4° C. After 3 washes in PBS,sections were successively incubated with the appropriate biotinylatedsecondary antibodies (anti human IgG, anti rabbit IgG dilution 1/125 inPBS, incubation 1 h at RT) and the avidin-HRP detection system (dilution1/125 in PBS, incubation 1 h at RT). The reaction was developed withdiaminobenzidine for exactly 15 min at RT. Tissue sections werecounterstained with hemalun for 1 min at RT, dehydrated in ascendingethanol series and coverslipped.

As shown in FIG. 8, a commercial TTR antibody Dako A0002 generated astrong staining in liver and intestine sections of FAP mice and did notproduce any stain in the corresponding TTR KO sections (FIG. 8 1A, 1B).The exemplary TTR antibody NI-301.35G11 generated a staining of similarpattern and intensity in both liver and intestine sections of FAP mice(FIG. 8 2A). In contrast, the exemplary antibody NI-301.37F1 generated astrong staining only in the intestine section but not on the liversection of FAP mouse (FIG. 8 3A). This indicates that the NI-301.37F1antibody binds only to the pathological (i.e. non-physiological) TTRaggregates that accumulate over time in the gastro-intestinal tract ofFAP mice, and not to TTR in native conformation as synthesized by theliver.

In addition to that, both exemplary antibodies NI-301.35G11 andNI-301.37F1 did not generate any staining in liver and intestine tissuesections from TTR-KO mice (FIG. 8 2B, 3B). In view of the high antibodyconcentrations used in this experiment with respect to the antibody'sbinding affinity, this absence of staining on TTR-KO sections isindicating high binding specificity for the TTR protein.

Example 8 Binding Selectivity for Misfolded, Misassembled and/orAggregated TTR Deposits in Human Tissue

The antibodies of the present invention were also evaluated for theircapacity to bind pathological TTR deposits in human tissue. Sections ofa skin biopsy from an FAP patient and sections of pancreas tissue from ahealthy individual were processed for immunohistochemistry using thesame procedure as described under Example 7, supra. Skin biopsy wasselected for this experiment as it contains an important amount ofpathological TTR amyloid deposits. In contrast, pancreas tissue was usedin this experiment because pancreatic alpha cells express TTR at highlevel.

As shown in FIG. 9, the commercial antibody Dako A0002 revealed anequally strong staining of pathological TTR deposits in the skin andnative TTR in pancreatic alpha cells (FIG. 9 1A). Similarly, theexemplary mouse chimeric antibody NI-301.mur35G11 produced an equallystrong staining of both pathological TTR deposits in the skin and nativeTTR in pancreatic alpha cells (FIG. 9 1B). In contrast, the antibodyNI-301.37F1 stained only the pathological TTR deposit in the skin andnot the native TTR in pancreatic alpha cells (FIG. 9 3A). This resultdemonstrates that NI-301.37F1 is highly selective by IHC forpathological TTR deposits, which include mutated, misfolded,misassembled and/or aggregated TTR conformations.

The “secondary antibody only” control condition presented in panels 1B,2B and 3B of FIG. 9 reveals the tissue staining that occurs in absenceof primary antibody. The absence (2B) or very low level (1B, 3B) ofstaining indicates that the staining observed in panels 1A, 2A and 3A isindeed specific for the corresponding primary antibodies.

Example 9 Assessment of the Binding Epitope of the TTR Antibodies

To determine the binding epitope of the exemplary antibodiesNI-301.59F1, NI301.35G11, and NI-301.37F1, the entire TTR amino acidsequence was analyzed using a panel of 29 sequential peptides 15 aminoacid long and 11 amino acid overlap, covalently bound to a membrane.Additional peptides including selected mutations were also plotted onthe membrane. The membrane was blocked in Roti blocking buffer overnightat 4° C., incubated first with the anti-TTR antibody diluted in blockingbuffer for 2 h at RT, then with an HRP-coupled anti human IgG antibodyfor 45 min at RT (dilution 1/20000 in TBS). The reaction was developedwith luminol and imaged by luminescence.

The antibody NI-301.59F1 recognizes the spots 15, 16 and 44 whichcorrespond to the sequence 61-EEEFVEGIY-69 (SEQ ID NO: 49) on full humanwild-type TTR, see FIG. 10 A. The antibody NI-301.35G11 recognizes thespots 13, 14, 42, and 44 which correspond to the sequence53-GELHGLTTEEE-63 (SEQ ID NO: 50) on full human wild-type TTR, see FIG.10 B. However, the antibody NI-301.35G11 does not recognize the spot 43,indicating this antibody cannot bind the sequence 53-GELHGPTTEEE-63corresponding to the L55P-TTR variant. The antibody

NI-301.37F1 recognizes the spots 9, 10, 11, 38, and 40 which correspondto the sequence 41-WEPFA-45 (SEQ ID NO: 51) on full human wild-type TTR,see FIG. 10C. However, the antibody NI-301.37F1 does not recognize thespot 43, indicating this antibody cannot bind the sequence 41-WGPFA-45corresponding to the E42G-TTR variant.

To refine determination of the binding epitope of the exemplaryantibodies NI-301.59F1, NI301.35G11, and NI-301.37F1, the entire TTRamino acid sequence was analyzed using a panel of 151 sequentialpeptides 15 amino acid long and 14 amino acid overlap, covalently boundto a membrane. For each peptide, the amino-acid in position 10 wasreplaced by an alanine for non-alanine amino-acids, whereas alanineswere replaced by glycine or proline. The membrane was blocked in Rotiblocking buffer overnight at 4° C., incubated first with the anti-TTRantibody diluted in blocking buffer for 2 h at RT, then with anHRP-coupled anti human IgG antibody for 45 min at RT (dilution 1/20000). The reaction was developed with luminol and imaged by luminescence.

The antibody NI-301.59F1 recognizes only the spots 77 and 83, indicatingthat E60 and V64 are not required for 59F1 binding whereas E61, E62,F63, E65, G66, 167 and Y68 are required for antibody binding. The exactcontribution of K69 is a matter of interpretation: strong antibodybinding to peptide 44 shown in FIG. 10A clearly indicates that absenceof K69 in C-terminal position does not prevent antibody binding; in thesubsequent experiment shown in FIG. 10E, however, K69A substitution inposition 10 on the peptide prevented antibody binding. These seeminglyopposite results suggest that NI-301.59F1 binds to a specificconformation of the amino-acid sequence 61-EEFXEGIY-68 (SEQ ID NO: 58),wherein X can be any amino acid.

The antibody NI-301.35G11 recognizes the spots 68, 71, 72, 73, 74 and75, indicating that G53 is not required for 35G11 binding whereas E54,L55, G57 and L58 are required for antibody binding. 35G11 bindingpattern also indicates that presence of E61 or E62 is required forantibody binding. The exact contribution of T59 and T60 could not bedetermined in this experiment, but it is hypothesized that the presenceof one of the two tyrosines is required for antibody binding. Takentogether, NI-301.35G11 binding profile on the alanine scan indicatesthat this antibody recognizes the sequence 54 ELXGLTXE 61 (SEQ ID NO:59), wherein X can encompassing all know amino acids, see FIG. 10F.

The antibody NI-301.37F1 binds to the spots 50, 52, 55, 56 and 58-62 onthe alanine scan membrane, and no to the spots 51, 53, 54 and 57. Thisindicates that W40, P42, F43 and A44 are required for antibody binding.Combined with the earlier observation that mutation E42G disruptsantibody binding (FIG. 10C), these results indicate that NI-301.37F1binds to the sequence 41-WEPFA-45 (SEQ ID NO: 60), see FIG. 10G.

Example 10 Determination of Antibody Binding Characteristics by SurfacePlasmon Resonance

The antibody binding characteristics to various soluble TTR preparationswere determined by means of surface plasmon resonance (SPR), using aBiorad Proteon XPR36 machine, see Table V.

SPR analysis was performed on a BioRad ProteOn XPR36 fitted with a GLMsensor chip. An anti-human antibody directed against the Fc gamma domainwas covalently coupled to the detection surface and saturated with theantibody under investigation. Wild-type and mutant TTR protein in nativeand misfolded conformations were diluted in HBS-T buffer atconcentrations ranging from 3.2 to 316 nM. The antibody-antigenassociation was analyzed during 180 s and the dissociation during 600 s.A Langmuir binding model (simple 1:1 association) was used to fit thedata and derive the association (ka)and dissociation (kd) constants, andthe affinity (KD).

An anti-human IgG-Fcy antibody was covalently coated on the detectionsurfaces, and used to capture the human TTR-specific antibodies. Theantibodies were probed with 4 different TTR preparations, includingnative and misfolded-aggregated wild-type TTR, and native V30M and L55PTTR mutants, all prepared at concentrations from 3.2 to 316 nM in HBS-Tbuffer (10 mM Hepes, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH7.4).Misfolded-aggregated wild-type TTR was prepared by acidic denaturationat 65° C. for 80 min in acetate buffer (50 mM acetate HCl, 100 mM KCl, 1mM EDTA, pH 3.0), with subsequent buffer exchange with HBS-T. 59F1,35G11 and 37F1 exhibited linear binding and dissociation characteristicswhich were best fitted with the Langmuir model.

The results show that these three antibodies bind with high affinity theV30M- and L55P-TTR variants in solution, as well as themisfolded-aggregated wild-type TTR preparation. In contrast, theseexemplary antibodies do not bind native wild-type TTR in solution.

Accordingly, the results show that NI-301.37F1 binds with high affinityto misfolded human wild-type TTR protein in solution, with a KD of 1.2nM, but not to the same protein in its native conformation. Similarbinding affinity (KD=1.4 nM) was measured for the mutant TTR-L55Pprotein.

TABLE V Determination of antibody binding characteristics by surfaceplasmon resonance. Antibody Langmuir fit (1:1 interaction model) NI-301.Antigen ka (M⁻¹s⁻¹) kd (s⁻¹) KD 59F1 native TTR n.a n.a >316 nMmisfolded- 9.7 10⁴ 3.4 10⁻⁴ 3.5 nM aggregated TTR native TTR- 1.3 10⁴2.2 10⁻⁴ 16 nM V30M native TTR- 5.1 10⁴ 1.5 10⁻⁴ 3.1 nM L55P 35G11native TTR-WT n.a n.a >316 nM misfolded- 2.3 10⁴ 2.7 10⁻⁴ 12 nMaggregated TTR native TTR- 7.4 10³ 2.4 10⁻⁴ 33 nM V30M native TTR- n.a.n.a. >100 nM L55P 37F1 native TTR-WT n.a n.a >316 nM misfolded- 2.1 10⁴2.6 10⁻⁵ 1.2 nM aggregated TTR native TTR- 1.1 10⁴ 1.9 10⁻⁴ 17 nM V30Mnative TTR- 3.3 10⁴ 4.6 10⁻⁵ 1.4 nM L55P

Example 11 Passive Immunization of Transgenic Mice for Human Va130MetTTR, Presenting Tissue TTR Deposition, with Chimeric Human-MouseRecombinant Anti-TTR Antibody Results in Removal of Deposition

Passive immunization was performed similar as described in internationalapplication WO 2010/030203, in particular Example 3, the disclosurecontent of which is incorporated herein by reference as well as of thereferences Kohno et al., Am. J. Pathol. (1997), 1497-1508 and Sousa etal., Am. J. Pathol. (2002), 1935-48, cited therein.

In brief, monoclonal antibody was administrated intraperitonealy weeklyfor 12 weeks at a dose of 3 mg/kg to 7-month-old and 17-month-old FAPmice, which were transgenic for human Va130Met-TTR allele and knockoutfor the murine TTR gene (Kohno et al., (1997), surpa). In the five daysfollowing the last dose, animals were sacrificed, and various tissueswere collected and fixed in paraformaldehyde solution, and embedded inparaffin. 3-5 μm sections were cut and processed forimmunohistochemistry using the commercial anti-TTR antibody describedabove. A standard immunofluorescence procedure was used, which was verysimilar to the one indicated in example 7 with only difference that afluorescent secondary antibody was used for detection. The surface oftissue invaded with TTR deposit was quantified and expressed aspercentage of total tissue area. Statistical analysis of treatmenteffect was performed with two-tailed, unpaired t-test.

This transgenic mouse line reproduce the key pathological mechanismcommon to TTR amyloid diseases which consists in TTR tetramerdisassembly and misfolding of the TTR monomers into a toxic andinsoluble amyloidogenic conformation. Like FAP patients, thesetransgenic mice typically present age-dependent TTR deposition. Theevaluation of treatment efficacy was investigated in two groups oftransgenic mice which were 7-month-old and 17-month-old at treatmentonset; ages where TTR deposition is important and invading manygastrointestinal tissues. Remarkably, passive immunization with theexemplary antibody NI-301.37F1 was associated with statisticallysignificant reduction in the tissue surface invaded with TTR depositionwhen treatment was started at 7 months of age, see FIG. 12A. Treatmenthad a similar effect in old mice, leading to almost significantreduction in TTR deposition, see FIG. 12B.

Example 12 Human-Derived, Recombinant Anti-TTR Antibodies Bind toPathological TTR Deposits In Vivo

To determine whether human-derived, recombinant anti-TTR antibodies areable to bind to pathological TTR deposits in vivo, adult FAP mice of 7months of age were injected with the antibody NI-301.37F1 at 30 mg/kgi.p. or with PBS for comparison. After 48 hours, these mice weresubmitted to transcardiac perfusion and tissues were processed forhistological analysis. Pathological TTR deposits were detected using arabbit polyclonal, anti-TTR antibody in combination with fluorescentlylabeled anti-rabbit IgG antibody, whereas the localization of theinjected antibody NI-301.37F1 was detected with a fluorescently labeledanti-human IgG antibody. In particular, immunoprecipitation wasperformed as follows.

Immunoprecipitation of NI-301.37F1 and isotype control antibodies frommouse plasma samples was performed for 2 hours at RT, using proteinA/G-coupled magnetic beads (Pierce #88803) loaded with anti-human IgGantibody (Jackson Immunoresearch #709-005-098). After 3 washes withPBS-T, samples were eluted from magnetic beads a 0.2M glycine buffer(pH2.5), neutralized with 1M Tris HCl (pH8.0), mixed with LDS-loadingbuffer (Life technologies #NP0007) and heated 10 min at 90° C. Sampleswere then loaded on a 4-12% bis-tris gel (Life technologies #WG1403A)run for 40 min at 200 V in MOPS running buffer. After protein transferon a nitrocellulose membrane, TTR protein was detected using either theconformation independent TTR antibody (Dako #A0002, 150 ng/ml) or theantibody NI-301.37F1 (20 nM), in combination with HRP-coupled protein A(Life technologies #10-1023, 1/10′000 dilution) and luminescent imaging.

The in vivo target engagement as described in FIG. 13 was performed inadult FAP mice (7-months-old) which received a single injection ofantibody NI-301.37F1 at 30 mg/kg i.p. or PBS. 48 hours later, mice wereperfused with PBS and organs were collected and processed forhistological analysis. Pathological TTR deposits were detected byimmunofluorescence using a commercial rabbit polyclonal anti-TTRantibody (Dako #A0002, 4.8 μg/ml) in combination with a Cy5-conjugatedanti-rabbit antibody (Jackson Immunoresearch #711-175-152, 1/200dilution). Presence (or absence) of NI-301.37F1 was detectedsimultaneously using a Cy3-conjugated anti-human antibody (JacksonImmunoresearch #709-165-149, 1/200 dilution). The same scanningparameters were used for imaging NI-301.37F1-injected and PBS-injectedtissues, and images received the same display adjustments.

As shown in FIG. 13, NI-301.37F1-dependent staining was highlycolocalized with TTR staining in NI-301.37F1-injected mice, but wascompletely absent in PBS-injected mice, as expected. This resultindicates that the anti-TTR antibody NI-301.37F1 is binding topathological TTR deposits in vivo.

Example 13 Detection of Misfolded TTR Protein Deposits In Vivo does notRequire Tissue Biopsies

This diagnostic procedure replacing tissue biopsy and histologicalanalysis in the diagnosis process of TTR amyloid diseases associatedwith aggregated, mutated, and/or misfolded TTR is exemplified hereinbelow and illustrated in FIG. 14. In particular, the experiment wasperformed with 7-month-old FAP mice, as described above; see Example 11,supra. These mice reproduce the core pathophysiological mechanism of FAPand, like patients, present age-dependent TTR deposition in varioustissues. Two FAP mice received a single intraperitonal injection of thehuman-derived, recombinant anti-TTR monoclonal antibody NI-301.37F1 at adose of 3 mg/kg. Prior antibody injection (t=0), and two days afterinjection (t=48 h), small blood samples were collected and plasma wereprepared for analysis. Plasma samples were submitted toimmunoprecipitation with an anti-human IgG antibody (to retrieve theinjected human anti-TTR antibody), with t=0 samples used as negativecontrols. The immunoprecipitation samples were then processed bywestern-blot to detect whether the anti-TTR antibody injected in micehad captured some misfolded TTR protein during the 48 hours where itcirculated in vivo. Western-blots were performed using bothconformation-specific and conformation-independent anti-TTR antibodies.A control experiment was performed with an isotype control antibody (notable to bind TTR protein) as negative control. An additional controlconsisted in incubating plasma samples from untreated FAP mice with theantibody NI-301.37F1 in vitro, and processing as described above.

The results presented in FIG. 14 indicate that antibody NI-301.37F1captured some misfolded TTR protein during the 48 hours incubationperiod in vivo. This was observed specifically for the antibodyNI-301.37F1 and not for the isotype control antibody. Furthermore, themisfolded TTR protein captured by antibody NI-301.37F1 was not presentin plasma samples collected from untreated mice. Altogether, theseresults indicate that the antibody NI-301.37F1 was able to removemisfolded TTR protein from insoluble TTR deposits in vivo, the presenceof which could be detected without the need for tissue biopsies. Onetechnical adjustment to use this diagnostic test in humans would consistin labeling the anti-TTR antibody allowing for its retrieval from humanplasma sample with, for example, a biotin or histidine or streptavidinetag. Alternatively, the unmodified anti-TTR antibody could be retrievedfrom human plasma sample by means of an anti-idiotypic antibody.

TABLE IV Mutations in the TTR gene Name (Protein Variant Reported incl.20-aa signal peptide) Sequence Variant (mRNA) Codon Change LocationPhenotype Ethnic Group References Gly6Ser (p.Gly26Ser) c.76G > A GGT >AGT Exon 2 non- Caucasian Jacobson (1994) Hum Mutat 3, 254 amyloidogenicCys10Arg (p.Cys30Arg) c.88T > C TGT > CGT Exon 2 AN, E, H, PN AmericanUemichi (1992) J Med Genet 29, 888 (Hungarian) Leu12Pro (p.Leu32Pro)c.95T > C CTG > CCG Exon 2 PN, AN, H, LM, L British Brett (1999) Brain122, 183 p.Met13Ile (p.Met33Ile) c.99G > C ATG > ATC Exon 2 non- GermanAltland (1999) The 4th International Symposium on FAP and Otheramyloidogenic TTR Related Disorders. Asp18Asn (p.Asp38Asn) c.112G > AGAT > AAT Exon 2 H American Connors (2003) Amyloid 10, 160 Asp18Gly(p.Asp 38Gly) c.113A > G GAT > GGT Exon 2 LM Hungarian Vidal (1996) Am JPathol 148, 361 Asp18Glu (p.Asp38Glu) c.114T > A or G GAT > GAA/G Exon 2PN South American Connors (2004) Amyloid 11, 61 Val20Ile (p.Val40Ile)c.118G > A GTC > ATC Exon 2 CTS, H German, American Jenne (1996) ProcNatl Acad Sci USA 93, 6302 Ser23Asn (p.Ser43Asn) c.128G > A AGT > AATExon 2 E, H, PN Portuguese, Connors (1999) Amyloid 6, 114 AmericanPro24Ser (p.Pro44Ser) c.130C > T CCT > TCT Exon 2 CTS, H, PN AmericanUemichi (1995) J Med Genet 32, 279 Ala25Ser (p.Ala45Ser) c.133G > TGCC > TCC Exon 2 H, PN American Yazatic (2002) Muscle Nerve 25, 244Ala25Thr (p.Ala45Thr) c.133G > A GCC > ACC Exon 2 CNS, PN JapaneseSekijima (2003) Lab Invest 83, 409 Val28Met (p.Val48Met) c.142G > AGTG > ATG Exon 2 PN Portuguese Carvalho (2000) Muscle Nerve 23, 1016Val30Leu (p.Val50Leu) c.148G > C GTG > CTG Exon 2 AN, H, K, PN Japanese,Murakami (1992) Biochem Biophys Res Commun 187, 397 American Val30Met(p.Val50Met) c.148G > A GTG > ATG Exon 2 AN, E, LM, PN American Chinese,Saraiva (1984) J Clin Invest 74, 104 Japanese, European Val30Ala(p.Val50Ala) c.149T > C GTG > GCG Exon 2 AN, H American Jones (1992)Clin Genet 41, 70 (German) Val30Gly (p.Val50Gly) c.149T > G GTG > GGGExon 2 CNS, E, LM American Peterson (1997) Ann Neurol 41, 307 Val32Ala(p.Val52Ala) c.155T > C GTG > GCG Exon 2 AN, H, PN Chinese Pica (2005)Muscle Nerve 32, 223 Val32Gly (p.Val52Gly) c.155T > G GTG > GGG Exon 2AN, PN French Plante-Bordeneuve (2003) J Med Genet 40, e120 Phe33Ile(p.Phe53Ile) c.157T > A TTC > ATC Exon 2 E, PN Jewish Jacobson (1988)Biochem Biophys Res Commun 153(1): 198 Phe33Leu (p.Phe53Leu) c.157T > CTTC > CTC Exon 2 PN American Li (1991) Neurology 41, 893 Phe33Val(p.Phe53Val) c.157T > G TTC > GTC Exon 2 PN Chinese, British Tachibana(1999) Amyloid 6(4): 282 Phe33Cys (p.Phe53Cys) c.158T > G TTC > TGC Exon2 CTS, E, K, A H American Connors (2003) Amyloid 10, 160 Arg34Gly(p.Arg54Gly) c.160A > G AGA > GGA Exon 2 E Kosovo Levy J, Hawkins PN,Rowczenio D, Godfrey T, Stawell R, Zamir E. The Ocular ImmunologyClinic, Royal Victorian Eye and Ear Hospital, Melbourne, Australia.Arg34Thr (p.Arg54Thr) c.161G > C AGA > ACA Exon 2 H, PN Italian Patrosso(1998) Am J Med Genet 77, 135 Lys35Asn (p.Lys55Asn) c.165G > C or TAAG > AAC/T Exon 2 AN, H, PN French Reilly (1995) Brain 118, 849Ala36Pro (p.Ala56Pro) c.166G > C GCT > CCT Exon 2 CTS, E, PN Greek,Italian, Jones (1991) Am J Hum Genet 48, 979 Jewish, American Asp38Ala(p.Asp58Ala) c.173A > C GAT > GCT Exon 2 AN, H, PN Japanese Yazaki(2000), Biochem Biophys Res Commun, 274(3): 702 Asp38Val (p.Asp58Val)c.173A > T GAT > GTT Exon 2 H, PN Guianese Lachmann (2002) N Engl J Med346, 1786 Asp39Val (p.Asn59Val) c.176A > T GAC > GTC Exon 2 H GermanEriksson (2009) Am J Surg Pathol 33 (1): 58 Trp41Leu (p.Trp61Leu)c.182G > T TGG > TTG Exon 2 E American Yazaki (2002) Amyloid 9, 263(Russian) Glu42Gly (p.Glu62Gly) c.185A > G GAG > GGG Exon 2 AN, H, PNJapanese, Russian, Ueno (1990) Biochem Biophys Res Commun 169, 1117American Glu42Asp (p.Glu62Asp) c.186G > C or T GAG > GAC/T Exon 2 HFrench Dupuy (1998) Amyloid 5, 285 Phe44Tyr (p.Phe64Tyr) c.191T > ATTT > TAT Exon 2 AN, PN French Plante-Bordeneuve (2003) J Med Genet 40,el20 Phe44Ser (p.Phe64Ser) c.191T > C TTT > TCT Exon 2 AN, H, PNAmerican Klein (1998) Neurology 51, 1462 Ala45Ser (p.Ala65Ser) c.193G >T GCC > TCC Exon 2 H Swedish Janunger (2000) Amyloid 7, 137 Ala45Thr(p.Ala65Thr) c.193G > A GCC > ACC Exon 2 H Irish, Italian, Saraiva(1992) Am J Hum Genet 50, 1027 American Ala45Asp (p.Ala65Asp) c.194C > AGCC > GAC Exon 2 H, PN Irish, American Saraiva (1995) Hum Mutat 5, 191Gly47Arg (p.Gly67Arg) c.199G > C GGG > CGG Exon 2 AN, PN JapaneseMurakami (1992) Biochem Biophys Res Commun 182, 520 Gly47Arg(p.Gly67Arg) c.199G > A GGG > AGG Exon 2 H, PN Italian Ferlini (2000)Clin Genet 57, 284 Gly47Ala (p.Gly67Ala) c.200G > C GGG > GCG Exon 2 AN,H, PN German, Italian, Ferlini (1994) Hum Mutat 4, 61 French Gly47Glu(p.Gly67Glu) c.200G > A GGG > GAG Exon 2 H, K, PN German, Italian Pelo(2002) Amyloid 9, 35 Gly47Val (p.Gly67Val) c.200G > T GGG > GTG Exon 2AN, CTS, H, PN Sri Lankan Booth (1993) Amyloid, 456 Thr49Ala(p.Thr69Ala) c.205A > G ACC > GCC Exon 3 CTS, H, PN Italian, FrenchAlmeida (1992) Hum Mutat 1, 211 Thr49Pro (p.Thr69Pro) c.205A > C ACC >CCC Exon 3 H, LM American Nakagawa (2008) J Neurol, 272 (1-2): 186;Connors (2003) Amyloid 10, 160; Thr49Ile (p.Thr69Ile) c.206C > T ACC >ATC Exon 3 H, PN Japanese Nakamura (1999) Hum Hered 49, 186 Thr49Ser(p.Thr69Ser) c.206C > G ACC > AGC Exon 3 PN Indian Rowczenio (2010) XIIInternational Symposium on Amyloidosis Ser50Ile (p.Ser70Ile) c.209G > TAGT > ATT Exon 3 AN, H, PN Japanese, Spanish Saeki (1992) FEBS Lett 308,35 Ser50Arg (p.Ser70Arg) c.210T > G AGT > AGG Exon 3 AN, H, PN Italian,French, Ueno (1990) Biochem Biophys Res Commun 169, 1117 JapaneseGlu51Gly (p.Glu71Gly) c.212A > G GAG > GGG Exon 3 H American Connors(2003) Amyloid 10, 160 Ser52Pro (p.Ser72Pro) c.214T > C TCT > CCT Exon 3AN, H, K, PN British Stangou (1998) Transplantation 66(2): 229 Gly53Glu(p.Gly73Glu) c.218G > A GGA > GAA Exon 3 CNS, LM, N French Ellie (2001)Neurology 57, 135 Gly53Ala (p.Gly73Ala) c.218G > C GGA > GCA Exon 3 AN,E, H, PN, LM British Douglass (2007) J Neurol Neurosurg Psychiatry 78,193 Glu54Leu (p.Glu74Leu) c.220_221 GA > TT GAG > TTG Exon 3 H BelgianRowczenio (2006) XI International Symposium on Amyloidosis Glu54Lys(p.Glu74Lys) c.220G > A GAG > AAG Exon 3 AN, H, PN Japanese Togashi(1999) Neurology 53, 637 Glu54Gly (p.Glu74Gly) c.221A > G GAG > GGG Exon3 AN, E, PN British Reilly (1995) Brain 118, 849 Glu54Asp (p.Glu74Asp)c.222G > T GAG > GAC Exon 3 Not listed German Eriksson (2009) Am J SurgPathol 33 (1): 58 Glu54Gln (p.Glu74Gln) c.220G > C GAG > CAG Exon 3 H,PN Romanian Coriu D, XIII International Symposium on AmyloisosisLeu55Gln (p.Leu75Gln) c.224T > A CTG > CAG Exon 3 AN, E, PN AmericanYazaki (2002) Amyloid 9, 268 (Spanish) Leu55Arg (p.Leu75Arg) c.224T > GCTG > CGG Exon 3 LM, PN German Connors (2003) Amyloid 10, 160 Leu55Pro(p.Leu75Pro) c.224T > C CTG > CCG Exon 3 AN, E, H, PN Taiwanese,Jacobson (1992) Hum Genet 89, 353 American (Dutch, German) His56Arg(p.His76Arg) c.227A > G CAT > CGT Exon 3 H American Jacobson (1999) TTRLocus-specific database Unpublished Leu58Arg (p.Leu78Arg) c.233T > GCTC > CGC Exon 3 AN, CTS, E, H Japanese Saeki (1991) Biochem Biophys ResCommun 180, 380 Leu58His (p.Leu78His) c.233T > A CTC > CAC Exon 3 CTS, HGerman, American Nichols (1989) Genomics 5, 535 (MD) Thr59Lys(p.Thr79Lys) c.236C > A ACA > AAA Exon 3 AN, H, PN Italian, AmericanSaraiva (1995) Hum Mutat 5, 191 (Asian) Thr60Ala (p.Thr80Ala) c.238A > GACT > GCT Exon 3 CTS, H, PN Australian, Wallace (1986) J Clin Invest 78,6 German, Irish, British, American Glu61Lys (p.Glu81Lys) c.241G > AGAG > AAG Exon 3 PN Japanese Shiomi (1993) Biochem Biophys Res Commun194, 1090 Glu61Gly (p.Glu81Gly) c.242A > G GAG > GGG Exon 3 CTS, H, PNAmerican Rosenzweig (2007) Amyloid 14, 65 (English/Dutch) Glu62Lys(p.Glu82Lys) c.243G > A GAG > AAG Exon 3 H Caucasian Briani C, CavallaroT, Ferrari S, Taioli F, Calamelli S, Verga L, Adami F, Fabrizi GM.Sporadic transthyretin amyloidosis with a novel TTR gene mutationmisdiagnosed as primary amyloidosis. J Neurol. 2012 Oct; 259(10):2226-8. Phe64Leu (p.Phe84Leu) c.250T > C TTT > CTT Exon 3 CTS, H, PNItalian, American Li (1991) Neurology 41, 893 Phe64Ser (p.Phe84Ser)c.251T > C TTT > TCT Exon 3 E, LM, PN, CNS Canadian (Italian), Uemichi(1999) Arch Neurol 56, 1152 British Gly67Glu (p.Gly87Glu) c.260G > AGGG > GAG Exon 3 H, PN Chinese Mak (2007) Amyloid, 14, 293 Ile68Leu(p.Ile88Leu) c.262A > T/C ATA > C/TTA Exon 3 H German, American Almeida(1991) Basic Res Cardiol 86, 567 Tyr69His (p.Tyr89His) c.265T > C TAC >CAC Exon 3 E Scottish, American Zeldenrust (1994) Amyloid, 1, 17Tyr69Ile (p.Tyr89Ile) c.265-266 TA > AT TAC > ATC Exon 3 CTS, H JapaneseTakei (2003) Amyloid 10, 25 Lys70Asn (p.Lys90Asn) c.270A > C/T AAA >AAC/T Exon 3 CTS, E, PN German, American Izumoto (1992) Neurology 42,2094 Val71Ala (p.Val91Ala) c.272T > C GTG > GCG Exon 3 CTS, E, PNFrench, Spanish Almeida (1993) Hum Mutat 2, 420. Ile73Val (p.Ile93Val)c.277A > G ATA > GTA Exon 3 AN, PN Bangladeshi Booth (1997) Hum Mutat12, 135 Asp74His (p.Asp94His) c.280G > C GAC > CAC Exon 3 non- GermanUemichi (1994) Amyloid, 1, 149 amyloidogenic Ser77Phe (p.Ser97Phe)c.290C > T TCT > TTT Exon 3 AN, PN French Plante-Bordeneuve (1998)Neurology 51, 708 Ser77Tyr (p.Ser97Tyr) c.290C > A TCT > TAT Exon 3 H,K, PN French, German, Wallace (1988) J Clin Invest 81, 189 American (IL,TX) Tyr78Phe (p.Tyr98Phe) c.293A > T TAC > TTC Exon 3 CTS, S, PN French(Italian) Magy (2003) Amyloid 10, 29 Ala81Thr (p.Ala101Thr) c.301G > AGCA > ACA Exon 3 H American Connors (2003) Amyloid 10, 160 Ala81Val(p.Ala101Val) c.302C > T GCA > GTA Exon 3 H Russian, Polish Rowczenio(2006) XI International Symposium on Amyloidosis Gly83Arg (p.Gly103Arg)c.307G > C GGC > CGC Exon 3 E Chinese Xie Y, Zhao Y, Zhou JJ, Wang X.Identification of a TTR gene mutation in a family with hereditaryvitreous amyloidosis Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2012 Feb;29(1): 13-5. Ile84Asn (p.Ile104Asn) c.311T > A ATC > AAC Exon 3 CTS, E,H American Skinner (1992) Ophthalmology 99, 503 Ile84Ser (p.Ile104Ser)c.311T > G ATC > AGC Exon 3 CTS, E, H, LM Hungarian, Swiss, Dwulet(1986) J Clin Invest 78, 880 American Ile84Thr (p.Ile104Thr) c.311T > CATC > ACC Exon 3 H, PN German, British Stangou (1998) Transplantation66, 229 Glu89Gln (p.Glu109Gln) c.325G > C GAG > CAG Exon 3 CTS, H, PNItaly Almeida (1992) Hum Mutat 1, 211 Glu89Lys (p.Glu109Lys) c.325G > AGAG > AAG Exon 3 AN, H, PN American Nakamura (2000) Amyloid 7, 46His90Asn (p.His110Asn) c.328C > A CAT > AAT Exon 3 non- German, Skare(1994) Clin Genet 45, 281 amyloidogenic Portuguese His90Asp(p.His110Asp) c.328C > G CAT > GAT Exon 3 H British Rowczenio (2006) XIInternational Symposium on Amyloidosis Ala91Ser (p.Ala111Ser) c.331G > TGCA > TCA Exon 3 AN, CTS, H, PN French Misrahi (1998) Hum Mutat 12, 71Gln92Lys (p.Gln112Lys) c.334G > A GAG > AAG Exon 3 H Japanese Saito(2001) Hum Pathol 32, 237 Val93Met (p.Val113Met) c.367G > A GTG > ATGExon 4 PN Malian Lozem (2008) The VIIth International Symposium on FAPand Other TTR Related Disorders. Val94Ala (p.Val114Ala) c.341T > C GTA >GCA Exon 4 AN, H, PN German, Greek Kristen (2007) Amyloid 14(4): 283(Cyprus) Ala97Ser (p.Ala117Ser) c.349G > T GCC > TCC Exon 4 PN, HChinese, Tachibana (1999) Amyloid 6, 282 Taiwanese Ala97Gly(p.Ala117Gly) c.350C > G GCC > GGC Exon 4 H, PN Japanese Yasuda (1994) JNeurol Sci 121, 97 Gly101Ser (p.Gly121Ser) c.361G > A GGC > AGC Exon 4non-amyloid Japanese Kishikawa M et al (1988) Hum Mutat 12, 363Pro102Arg (p.Pro122Arg) c.365C > G CCC > CGC Exon 4 non-amyloid GermanAltland (1999) The 4th International Symposium on FAP and Other TTRRelated Disorders. Arg103Ser (p.Arg123Ser) c.367C > A CGC > AGC Exon 4 HAmerican Connors (2003) Amyloid 10, 160 Arg104Cys (p.Arg124Cys) c.370C >T CGC > TGC Exon 4 non-amyloid, American Torres (1996) NeuromuscularDisordVol 6, S21, Arg104His (p.Arg124His) c.371G > A CGC > CAC Exon 4non-amyloid Japanese, Terazaki (1999) Biochem Biophys Res Commun 264,365 American Ile107Val (p.Ile127Val) c.379A > G ATT > GTT Exon 4 CTS, H,PN German, American Jacobson (1994) Hum Mutat 3, 399 Ile107Phe(p.Ile127Phe) c.379A > T ATT > TTT Exon 4 AN, PN British Rowczenio(2006) XI International Symposium on Amyloidosis Ile107Met (p.Ile127Met)c.381T > G ATT > ATG Exon 4 H, PN German Connors (2003) Amyloid 10, 160Ala108Ala (p.Ala128Ala) c.384C > T GCC > GCT Exon 4 non- PortuguesePalha (1997) Amyloid 4, 52 amyloidogenic Ala109Ser (p.Ala129Ser)c.385G > T GCC > TCC Exon 4 PN Japanese Date (1997) J Neurol Sci 150,143 Ala109Thr (p.Ala129Thr) c.385G > A GCC > ACC Exon 4 non- PortugueseMoses (1990) J Clin Invest 86, 2025 amyloidogenic Ala109Val(p.Ala129Val) c.386C > T GCC > GTC Exon 4 non- American Izumoto (1993) JRheumatol 20 188 amyloidogenic Leu111Met (p.Leu131Met) c.391C > A CTG >ATG Exon 4 CTS, H Danish Nordlie (1988) Scand J Immunol 27, 119Ser112Ile (p.Ser132Ile) c.395G > T AGC > ATC Exon 4 H, PN Italian DeLucia (1993) Clin Neuropathol 12, S44 Tyr114His (p.Tyr134His) c.400T > CTAC > CAC Exon 4 CTS Japanese Murakami (1994) Neurology 44, 315Tyr114Cys (p.Tyr134Cys) c.401A > G TAC > TGC Exon 4 AN, E, H, LM, PNJapanese Ueno (1990) Biochem Biophys Res Commun 169, 143 Tyr116Ser(p.Tyr136Ser) c.407A > C TAT > TCT Exon 4 AN, PN, CTS French Misrahi(1997) Hum Mutat 12, 71 Thr119Met (p.Thr139Met) c.416C > T ACG > ATGExon 4 non- Portuguese, Harrison (1991) Am J Med Genet 39, 442amyloidogenic American Ala120Ser (p.Ala140Ser) c.418G > T GCT > TCT Exon4 AN, H, PN Caribbean Lachmann (2002) N Engl J Med 346, 1786 Val122del(p. Val142del) c.424_426 del GTC Exon 4 CNS, CTS, H, PN American Uemichi(1997) Neurology 48 (Ecuador/Spain) Val122Ile (p.Val142Ile) c.424G > AGTC > ATC Exon 4 H African, Jacobson (1990) Am J Hum Genet 47, 127Portuguese, American Val122Ala (p.Val142Ala) c.425T > C GTC > GCC Exon 4E, H, PN British, American Theberge (1999) Amyloid 6, 54 Pro125Ser(p.Pro145Ser) c.433C > T CCC > TCC Exon 4 non- Italian Ferlini (1996)Neuromuscular Disord Vol 6, S23, amyloidogenic Abbreviation Key: AN =autonomic neuropathy; CTS = carpal tunnel syndrome; E = eye; H = heart,K = kidney, L = liver, LM = leptomeningeal; N = neuropathy; PN =polyneuropathy; CNS = central nervous system

1. A human-derived anti-transthyretin (TTR) antibody or anantigen-binding fragment thereof.
 2. The antibody of claim 1, wherein atleast the complementarity determining regions (CDRs) and/or variableregion are encoded by a cDNA derived from an mRNA obtained from a humanmemory B cell which produced an anti-TTR antibody.
 3. The antibody ofclaim 1 or 2, which is capable of binding mutated, misfolded,misassembled and/or aggregated TTR species and/or fragments thereof anddoes not substantially recognize physiological TTR species.
 4. Theantibody of any one of claims 1 to 3, wherein the antibody is capable ofbinding (i) a TTR epitope which comprises or consists of the amino acidsequence EEEFVEGIY (SEQ ID NO: 49), GELHGLTTEEE (SEQ ID NO: 50), orWEPFA (SEQ ID NO: 51), and/or (ii) a non-linear, conformational epitope.5. The antibody of claim 4, wherein the antibody binds the TTR epitope(i) GELHGLTTEEE (SEQ ID NO: 50) but not a corresponding L55P mutantepitope or (ii) WEPFA (SEQ ID NO: 51) but not a corresponding E42Gmutant.
 6. The antibody of any one of claims 1 to 5 or a TTR bindingmolecule comprising in its variable region or binding domain (a) atleast one complementarity determining region (CDR) of the V_(H) and/orV_(L) variable region amino acid sequence depicted in any one of FIGS.1A to 1T; (b) an amino acid sequence of the V_(H) and/or V_(L) region asdepicted in any one of FIGS. 1A to 1T; (c) at least one CDR consistingof an amino acid sequence resulted from a partial alteration of any oneof the amino acid sequences of (a); or (d) a heavy chain and/or a lightvariable region comprising an amino acid sequence resulted from apartial alteration of the amino acid sequence of (b).
 7. The antibody ofclaim 6, wherein the antibody or antigen binding fragment thereoffurther comprises a polypeptide sequence which is heterologous to the VHand/or VL region or the least one CDR, preferably wherein polypeptidesequence comprises a constant domain
 8. The antibody of claim 7, whereinthe constant domain is a human constant domain, preferably of the IgGtype, most preferably of the IgG1 class or isotype.
 9. An anti-TTRantibody or TTR-binding molecule which binds to the same epitope as theantibody of claim
 6. 10. The antibody of any one of claims 5 to 9, whichis a chimeric murine-human or a murinized antibody.
 11. The antibody ofany one of claims 1 to 10, which is an antibody fragment selected fromthe group consisting of a single chain Fv fragment (scFv), an F(ab′)fragment, an F(ab) fragment, and an F(ab′)₂ fragment.
 12. Apolynucleotide encoding the antibody of any one of claims 1 to 11,preferably wherein the polynucleotide is a cDNA
 13. A vector comprisingthe polynucleotide of claim
 12. 14. A host cell comprising thepolynucleotide of claim 12 or a vector of claim
 13. 15. Use of thepolynucleotide of claim 12, the vector of claim 13, or the host cell ofclaim 14 for the production of an anti-TTR antibody.
 16. A method forpreparing an anti-TTR antibody or a biotechnological derivative thereof,said method comprising (a) culturing the cell of claim 14; and (b)isolating the antibody from the culture.
 17. An antibody encoded by apolynucleotide of claim 12 or obtainable by the method of claim
 16. 18.The antibody of any one of claim 1 to 11 or 17, which (i) comprises adetectable label, preferably wherein the detectable label is selectedfrom the group consisting of an enzyme, a radioisotope, a fluorophoreand a heavy metal; and/or (ii) is attached to a drug.
 19. A peptidehaving an epitope of TTR specifically recognized by an antibody of anyone of claim 1 to 11, 17 or 18, preferably wherein the peptide comprisesan amino acid sequence as defined in claim 4 or a modified sequencethereof in which one or more amino acids are substituted, deleted and/oradded, wherein the peptide is recognized by the antibody of any one ofclaims 4 to 6, preferably wherein the peptide is an antigen.
 20. Acomposition comprising the antibody of any one of claim 1 to 11, 17 or18, the polynucleotide of claim 12, the vector of claim 13, the cell ofclaim 14 or the peptide of claim 19, preferably wherein the composition(i) is a pharmaceutical composition and further comprises apharmaceutically acceptable carrier, preferably wherein the compositionis a vaccine and/or comprises an additional agent useful for preventingor treating diseases associated with TTR amyloidosis; or (ii) adiagnostic composition, preferably further comprising reagentsconventionally used in immuno or nucleic acid based diagnostic methods.21. An anti-TTR antibody or a TTR-binding fragment of any one of claim 1to 11, 17 or 18 having substantially the same binding specificities ofany one thereof, the polynucleotide of claim 12, the vector of claim 13,the cell of claim 14, the peptide of claim 19 or the composition ofclaim 20 for use in the prophylactic or therapeutic treatment of adisease associated with TTR amyloidosis, preferably wherein the diseaseis selected from the group consisting of Familial Amyloid Polyneuropathy(FAP), Familial Amyloid Cardiomyopathy (FAC), Senile SystemicAmyloidosis (SSA), systemic familial amyloidosis, leptomeningeal/CentralNervous System (CNS) amyloidosis including Alzheimer disease,TTR-related ocular amyloidosis, TTR-related renal amyloidosis,TTR-related hyperthyroxinemia, TTR-related ligament amyloidosisincluding carpal tunnel syndrome, rotator cuff tears and lumbar spinalstenosis, and preeclampsia.
 22. A TTR-binding molecule comprising atleast one CDR of an antibody of any one of claim 1 to 11, 17 or 18 foruse in in vivo detection or imaging of or targeting a therapeutic and/ordiagnostic agent to TTR in the human or animal body, preferably whereinsaid in vivo imaging comprises scintigraphy, positron emissiontomography (PET), single photon emission tomography (SPECT), nearinfrared (NIR), optical imaging or magnetic resonance imaging (MRI). 23.An in vitro method for diagnosing a disorder associated with TTRamyloidosis, comprising steps of determining the presence of an antibodythat binds to a peptide of claim 19 in a biological sample of saidsubject.
 24. A kit useful in the diagnosis or monitoring of a disorderassociated with TTR amyloidosis, said kit comprising at least oneantibody of any one of claim 1 to 11, 17 or 18 or a TTR binding moleculehaving substantially the same binding specificities of any one thereof,the polynucleotide of claim 12, the vector of claim 13 or the cell ofclaim 14 and/or the peptide of claim 19, optionally with reagents and/orinstructions for use.
 25. A method of diagnosing a disease associatedwith TTR amyloidosis as defined in claim 21, monitoring the treatment ofthe disease with an anti-TTR antibody or determining the diagnostic ortherapeutic utility of an anti-TTR antibody comprising assaying thelevel of misfolded and/or aggregated TTR in a sample of a body fluidfrom a subject following administration of an anti-TTR antibody to thesubject, wherein the presence or elevated the level of misfolded and/oraggregated TTR in the sample of the subject compared to a controlindicates a disease associated with TTR amyloidosis.
 26. The method ofclaim 25, wherein the level of misfolded and/or aggregated TTR in thesample is assayed by determining a complex formed between the anti-TTRantibody and the misfolded and/or aggregated TTR.
 27. The method ofclaim 25 or 26, wherein the control is a sample obtained from thesubject prior to administration of the anti-TTR antibody.
 28. The methodof any one of claims 25 to 27, wherein the body fluid is blood and thesample is derived thereof and/or the control is a corresponding sampleof a body fluid taken from the subject prior to administration of theanti-TTR antibody.
 29. The method of any one of claims 25 to 28, whereinthe subject is a non-human animal or a human
 30. The method of any oneof claims 25 to 30, wherein the sample from the subject is obtained at aspecified time interval following administration.
 31. The method ofclaim 30, wherein the time interval is less than a week, preferably lessthan or equal to 48 hours.
 32. The method of any one of claims 25 to 31,wherein the anti-TTR antibody is an anti-TTR antibody or antigen-bindingfragment of any one of claim 1 to 11, 17 or
 18. 33. Use of an anti-TTRantibody in the method of any one of claims 25 to 32, preferably whereinthe antibody is an anti-TTR antibody or antigen-binding fragment of anyone of claim 1 to 11, 17 or 18.